Antagonistic dr3 ligands

ABSTRACT

The present disclosure relates to treatment of inflammatory diseases. In particular, the present disclosure relates to antagonistic DR 3  ligands useful for treating inflammatory diseases.

BACKGROUND

TL1A is a TNF-superfamily member produced by endothelial cells,dendritic cells, monocytes and other immune cells. TL1A signals throughDR3—a TNF receptor-superfamily member expressed by activated T-cells andother immune cells. Receptor ligation by TL1A leads to increasedproliferation and cytokine production by T-helper effector cells. DR3and TL1A are involved in RA and CD and antagonizing the DR3-inducedeffects would therefore be desirable in treatment of inflammatorydiseases such as e.g. RA (Rheumatoid Arthritis) and CD (Crohns Disease).

WO2011106707 discloses a DR3 specific antibody (11H08), as well asvariants thereof, comprising the 11H08 CDR sequences (SEQ ID NO 14+15)inserted into various antibody frameworks. The 11H08 antibody binds DR3with a relatively low affinity and it does not bind to the CRD1 domain.There is thus a need in the art for DR3 antagonists useful for treatinginflammatory diseases.

SUMMARY

Bivalent antibodies raised against DR3 have agonistic effects. A few ofthese agonistic DR3 specific antibodies have the ability to blockinteraction between DR3 and TL1A. As agonistic antibodies lead toincreased proliferation and cytokine production by T helper effectorcells, it is undesirable to use bivalent DR3 antibodies in connectionwith treatment of inflammatory disorders.

The present invention provides antagonistic DR3 ligands, wherein saidligands have a monovalent specificity for DR3, and wherein said ligandblocks binding of TL1A to DR3. Such ligands are preferably derived froma bivalent agonistic antibody and they are optionally conjugated with ahalf life extending moiety such as e.g. a lipophilic moiety. Suchligands preferably have a high affinity, and/or preferably bind to theCRD1 domain of DR3. The present invention furthermore relates to use ofsuch ligands for treating inflammatory diseases. The DR3 ligands of thepresent invention are shown herein to be capable of antagonizing effectsinduced via DR3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The sequences referred to herein.

FIG. 2: DR3 expression on CHOK1SV analysed by FCM. JD3 is a commerciallyavailable DR3 antibody.

FIG. 3: Stable DR3 expression on CHOK1SV cultured with 100 μM MSX.Expression analysed by FCM.

FIG. 4: Sequence alignment of TNFR1 and DR3 extracellular domains. Eachline represents a cysteine rich domain (CRD) which again can be dividedinto A and B sub-domains. The conserved disulfide pattern in CRD, asdetermined for TNFR1, is highlighted.

FIG. 5: Individual mice-sera pre-screened for the ability to blockhumanTL1A binding to CHO cells over-expressing DR3. The right bar(black) is anti-TL1A control (MAB7441 RnD Biosystems).

FIG. 6: Examples of antibodies blocking the TL1A:DR3 interaction done byflow cytometry. FIG. 6A show specific binding to DR3 cells of 6 positiveDR3 antibodies. FIG. 6B show inhibition studies showing 4 blockingantibodies and two non-blockers. Y-axis shows mean intensityfluorescence.

FIG. 7: FIG. 7A Titration curves for 3 of the blocking antibodies andnon-blocking DR3 specific ab as control—shown with full antibodies andwith Fab's.

FIG. 8: CD4+ T cells stimulated with IL12/IL18+TL1A with and withoutanti-DR3Fabs vs mAb. T cell proliferation is measured on day 5.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention realized that production of theDR3 antigen—soluble as well as cell-surface expressed versionsthereof—proved difficult as none of the traditional approaches weresuccessful. Recombinant expression of the extracellular domain of DR3 inhuman cell lines usually led to the secretion of soluble proteins thatcontained large amounts of oligomers and high molecular weight complexes(see also example 3). These oligomerized protein batches were presumablynot optimal for immunizations. In parallel with soluble proteinexpression optimization (as described in example 3), mice were immunizedwith cells over-expressing membrane-bound DR3. Production of a stablecell line over-expressing DR3 was, however, not straight-forward. Thedeath domain in full length DR3 leads to cell-death in stablytransfected cell lines over-expressing DR3 and it was thereforenecessary to modify the full length DR3 (see examples 2 and 5).Immunizations have been performed in different mice strains (BALB/C, RBFand NMRCF1) in order to increase the antibody-repertoire diversity andthe likelihood of generating neutralizing anti-DR3 Abs.

Several hundred DR3-binding antibodies were identified; of these onlyfew (˜2%) were able to block/inhibit DR3:TL1A binding. The DR3antibodies with the ability to block DR3:TL1A binding were thus presumedto have the ability to antagonize DR3 induced effects. It did, however,turn out that all DR3 antibodies—regardless of whether they had theability to block DR3:TL1A binding or not—were agonistic both in thepresence and absence of TL1A, i.e. they did apparently to some extentmimic the effects on DR3 that TL1A binding induces.

Based on these surprising observations, the inventors hypothesized thatthe explanation for the agonistic effects exerted by all the DR3antibodies could possibly be that any bivalent DR3 antibody would resultin clustering of DR3 and that DR3 clustering might have the potential toelicit intracellular DR3 signaling. This hypothesis furthermore findssupport in recent publications regarding TNFR family members Fas (CD95)and TNFR2 (Wang et al. (2010) Nature Struc. Mol. Biol. 17, 1324-1328;Mukai et al. (2010) Sci. Signal. 3, ra83). Wang et al provide bothstructural data and solution data demonstrating that the intracellularsignaling complex are of higher order and contains at least 5-7 copiesof the receptor. Similarly, Mukai et al demonstrate that clustering ofthe extracellular part of the receptor is induced by ligand binding.Thus, both publications indicate that higher order clustering of theseTNFR family members may be a prerequisite for signaling.

In order to test this hypothesis, Fab fragments (monovalent DR3antibodies) produced by cleavage of mAb's by papain were tested infunctional assays. The surprising outcome from these assays was thatmonovalent DR3 antibodies (made on basis of the DR3 antibodies havingthe ability to block/inhibit DR3:TL1A binding) were antagonistic infunctional assays, i.e. they had the ability to inhibit DR3 inducedeffects. Monovalent DR3 ligands/antibodies do therefore not facilitateDR3-clustering and they do therefore not have agonistic effects.

Antibodies that did not prevent TL1A:DR3 interaction were used asnegative controls. This type of antibodies are agonistic in the absenceof TL1A at very high concentrations but only as mAbs. The correspondingFabs from these antibodies were not able to prevent TL1A-inducedeffects.

DEFINITIONS

“Inflammation” is the complex biological response of vascular tissues toharmful stimuli, such as pathogens, damaged cells, or irritants.Inflammation is a protective attempt by the organism to remove theinjurious stimuli as well as initiate the healing process for thetissue. Inflammation is not a synonym for infection—infection is causedby an exogenous pathogen, while inflammation is the responses of theimmune system in the organism to the pathogen.

Normally, the immune system is able to distinguish between the body'snormal cells or “self” and foreign pathogens or abnormal cells or“non-self”. The process by which the immune system loses the ability torecognize “self” as normal and the subsequent response directed againstthe tissue or cells, results in loss of tolerance, a state of“autoimmunity”. The pathologies resulting from autoimmunity often haveserious clinical consequences and are one of the major health problemsin the world, especially in developed nations.

Biologic therapeutics are now available for the treatment of certainautoimmune diseases and/or cancer. For example, patients with rheumatoidarthritis may be treated with Rituximab (anti-CD20), and patients withCrohn's disease may be treated with Infliximab or Natalizumab.Unfortunately, patients that receive treatment with any one of thesebiologics also experience a variety of side-effects and/or arenon-responders and/or develop inhibitors. There is still a need foralternative biological medicaments which specifically targetpathological tissue and/or which do not affect healthy tissue and/orwhich result in less severe side effects and/or which result in fewerside effects and/or which may be used long-term and/or which do notresult in the formation of inhibitors. The current invention relates tothese unmet needs amongst patients with autoimmune diseases and in thosewith chronic inflammatory diseases.

The ligands of the present invention are thus suitable for use intreatment of inflammatory diseases and conditions such as e.g.psoriasis, type I diabetes, Grave's disease, Inflammatory bowel disease(IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome,multiple sclerosis, rheumatoid arthritis (RA), autoimmune myocarditis,Kawasaki disease, coronary artery disease, chronic obstructive pulmonarydisease, interstitial lung disease, autoimmune thyroiditis, systemiclupus erythematosus (SLE), scleroderma, systemic sclerosis, psoriaticarthritis, osteoarthritis, atoptic dermatitis, vitiligo, graft vs. hostdisease, Sjöogrens's syndrome, autoimmune nephritis, Goodpasture'ssyndrome, chronic inflammatory demyeliniating polyneutopathy, allergy,asthma and other autoimmune diseases.

Crohn's disease (CD/granulomatous/colitis) is an inflammatory disease ofthe intestines that may affect any part of the gastrointestinal tractfrom mouth to anus, causing a wide variety of symptoms. It primarilycauses abdominal pain, diarrhea (which may be bloody), vomiting, orweight loss, but may also cause complications outside of thegastrointestinal tract such as skin rashes, arthritis, inflammation ofthe eye, tiredness, and lack of concentration. There is no knownpharmaceutical or surgical cure for Crohn's disease. Treatment optionsare restricted to controlling symptoms, maintaining remission andpreventing relapse.

Rheumatoid Arthritis (RA):

RA is a systemic disease that affects the entire body and is one of themost common forms of arthritis. It is characterized by the inflammationof the membrane lining the joint, which causes pain, stiffness, warmth,redness and swelling. Inflammatory cells release enzymes that may digestbone and cartilage. As a result of rheumatoid arthritis, the inflamedjoint lining, the synovium, can invade and damage bone and cartilageleading to joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds.

“DR3” is sometimes referred to as Death Receptor 3, TRAMP, TNFRSF12,TNFR25, TNFRS25, APO-3, DDR3, LARD, TR3, WSL-1, or WSL-LR. Human DR3 isa member of the TNF receptor (TNFR) super family comprising fourcysteine-rich motives in the extracellular domain and a “death domain”in the cytoplasmic domain. Human DR3 comprises the amino acid sequenceas defined in SEQ ID 1. The extracellular domain of DR3 (residues25-199) comprises four cysteine-rich domains (CRD1, CRD2, CRD3 andCRD4). Each CRD typically contains six cysteine residues that form threedisulfide bounds. In addition each CRD can be subdivided into modules A1and B2 which are typically observed in conventional members of the TNFRsuperfamily.

“Block/inhibit/reduce binding of DR3 to TL1A”. Monovalentligands/antibodies according to the present invention have the abilityto inhibit/block/reduce DR3:TL1A binding. It can be tested in ahigh-throughput image based assay. This was done in an FMAT system, byscreening for the ability to bind DR3 transfected CHO cells andcounter-screened against wild-type cells (described more in detail inexample 4). Monovalent ligands/antibodies according to the presentinvention have the capacity to block or inhibit or reduce DR3:TL1Abinding, as measured in this assay, if DR3:TL1A binding is reduced atleast 10%, preferably at least 20%, preferably at least 25% preferablyat least 30%, preferably at least 40%, preferably at least 50%,preferably at least 60%, preferably at least 70%, preferably at least75%, preferably at least 80%, preferably at least 90%, preferably atleast 95% and most preferably about 100%.

“Protractive groups”/“half life extending moieties” is herein understoodas one or more chemical groups attached to one or more amino acid sitechain functionalities such as —SH, —OH, —COOH, —CONH₂, —NH₂, or one ormore N- and/or O-glycan structures and that can increase in vivocirculatory half life of a number of therapeutic proteins/peptides whenconjugated to these proteins/peptides. Examples of protractivegroups/half life extending moieties include but not limited to are:Biocompatible fatty acids and derivatives thereof, Hydroxy Alkyl Starch(HAS) e.g. Hydroxy Ethyl Starch (HES), Poly Ethylen Glycol (PEG), Poly(Gly_(x)-Ser_(y))_(n) (HAP), Hyaluronic acid (HA), Heparosan polymers(HEP), Phosphorylcholine-based polymers (PC polymer), Fleximers,Dextran, Poly-sialic acids (PSA), an Fc domain, Transferrin, Albumin,Elastin like peptides, XTEN polymers, Albumin binding peptides, a CTPpeptide, and any combination thereof.

“PEGylated DR3 ligand variants” according to the present invention mayhave one or more PEG molecule attached to any part of the DR3 ligandpolypeptide including any amino acid residue or carbohydrate moiety ofthe DR3 ligand polypeptide. Chemical and/or enzymatic methods can beemployed for conjugating PEG or other protractive groups to a glycan onthe monovalent DR3 ligand according to the invention. An example of anenzymatic conjugation process is described e.g. in WO03031464. Theglycan may be naturally occurring or it may be inserted via e.g.insertion of an N-linked glycosylation site using methods well known inthe art. “Cysteine-PEGylated DR3 ligand variant” according to thepresent invention have one or more PEG molecules conjugated to asulfhydryl group of a cysteine present in the DR3 ligand.“Cysteine-acylated DR3 ligand variant” or “Cysteine-alkylated DR3 ligandvariant” according to the present invention have one or more hydrophobichalf life extending moieties conjugated to a sulfhydryl group of acysteine introduced in the DR3 ligand. It is furthermore possible tolink protractive half life extending moieties to other amino acidresidues.

The most abundant protein component in circulating blood of mammalianspecies is serum albumin, which is normally present at a concentrationof approximately 3 to 4.5 grams per 100 milliliters of whole blood.Serum albumin is a blood protein of approximately 65,000 daltons whichhas several important functions in the circulatory system. It functionsas a transporter of a variety of organic molecules found in the blood,as the main transporter of various metabolites such as fatty acids andbilirubin through the blood, and, owing to its abundance, as an osmoticregulator of the circulating blood. Serum albumin has a half-life ofmore than one week, and one approach to increasing the plasma half-lifeof proteins has been to conjugate to the protein a moiety that binds toserum albumin. Albumin binding property may be determined as describedin J. Med. Chem., 43, 1986, (2000) which is incorporated herein byreference.

Hydrophobic/Lipophilic Half Life Extending Moiety:

The ligands according to the present invention are preferably conjugatedwith a half life extending moiety that is largely lipophilic/hydrophobicin nature. In a preferred embodiment, the hydrophobic half lifeextending moiety is capable of forming non-covalent complexes withalbumin (“albumin binder”), thereby promoting the circulation of thederivative with the blood stream, and also having the effect ofextending the time of action of the derivative. Thus, a preferredsubstituent, or moiety, as a whole may be referred to as an albuminbinding moiety.

The half life extending moiety is preferably at, or near, the oppositeend of the albumin binding moiety as compared to its point of attachmentto the DR3 ligand according to the invention. The other portion of thealbumin binding moiety, i.e. the portion in-between the half lifeextending moiety and the point of attachment to the peptide, may bereferred to as a linker moiety, linker, spacer, or the like. However,the presence of a linker is optional, and hence the albumin bindingmoiety may be identical to the half life extending moiety.

In particular embodiments, the albumin binding moiety and/or the halflife extending moiety is lipophilic, and/or negatively charged atphysiological pH (7.4).

The albumin binding moiety and/or the half life extending moiety may becovalently attached to an amino group of the peptide by conjugationchemistry such as by alkylation, acylation, or amide formation; or to ahydroxyl group, such as by esterification, alkylation; or to othergroups through oximation.

In a preferred embodiment, an active thiophilic derivative of thealbumin binding moiety and/or the half life extending moiety iscovalently linked to the thiol of a cysteine residue of the anti-DR3Fab.Such thiophilic groups include, but are not limited to, maleimides,halo-maleimides, halides (especially α-haloacetyl), acryloyl-derivatives(eg. acrylates and acrylamides), vinylsulfones, reactive disulfidegroups (eg. 2-pyridyl). Thus, the anti-DR3Fab′ of the present inventionis preferably linked to the albumin binding moiety through a thioetheror disulfide bond.

Monovalent antibodies according to the present invention, such as e.g.Fab′ fragments, may be designed to contain the naturally occurringcysteine residue from the heavy chain that forms part of one of theheavy chain sulphur bridges of an intact antibody. This cysteine residueis termed C239 (Kabat numbering). Cysteine residues can also be insertedby genetic engineering but there may be safety advantages associated byemploying naturally occurring cysteine residues for conjugationpurposes.

In a preferred embodiment, an active ester of the albumin binding moietyand/or the hydrophobic half life extending moiety is covalently linkedto an amino group of a sialic acid residue or a sialic acid derivative,under formation of an amide bond (this process being referred to asacylation).

According to a highly preferred embodiment of the present invention, thealbumin binding moiety is attached to the ligand via a glycan usingenzymatic methods such as e.g. a method involving use of asialilyltransferase.

For the present purposes, the terms “albumin binding moiety”, “half lifeextending moiety”, and “linker” include the un-reacted as well as thereacted forms of these molecules. Whether or not one or the other formis meant is clear from the context in which the term is used.

The term “fatty acid” refers to aliphatic monocarboxylic acids havingfrom 4 to 28 carbon atoms, it is preferably unbranched, and/or evennumbered, and it may be saturated or unsaturated

The term “fatty diacid” refers to fatty acids as defined above but withan additional carboxylic acid group in the omega position. Thus, fattydiacids are dicarboxylic acids.

The nomenclature is as is usual in the art, for example —COOH, as wellas HOOC—, refers to carboxy; —C₆H₄— to phenylen; —CO—, as well as —OC—,to carbonyl (O═C<); C₆H₅—O— to phenoxy; and halide refers to thehalogens —F, —Cl, —Br, —I, and —At.

In a preferred embodiment, the albumin binding moiety of the presentinvention comprises a fatty acyl group (—(CH₂)_(n)—CO—, where n=1, 2, 3,. . . 40) or an omega-carboxy fatty acyl group (HO₂C—(CH₂)_(n)—CO—,where n=1, 2, 3, . . . 40) linked to the peptide or protein via a linkerand a sialic acid residue or sialic acid derivative.

In a preferred embodiment, the albumin binding moiety of the presentinvention comprises a fatty acyl group (—(CH2)_(n)—CO—, where n=1, 2, 3,. . . 40) or an omega-carboxy fatty acyl group (HO₂C—(CH₂)_(n)—CO—,where n=1, 2, 3, . . . 40) linked to the peptide or protein via a linkerand a cysteine residue. In a particular preferred embodiment, n is 16 or18.

In another preferred embodiment, the albumin binding moiety of thepresent invention comprises a fatty acyl group of the typeR—(CH2)_(n)—CO—, where n=1, 2, 3, . . . 40, linked to the peptide orprotein via a linker and a cysteine residue. R is a group comprising anacidic group, eg. tetrazol-5-yl or —O—C6H4-COOH. In a particularpreferred embodiment, n is 14 or 15.

Compounds having a —(CH₂)₁₂— moiety are possible albumin binders in thecontext of this invention. If such a compound is attached to a proteinor peptide and results in an increased plasma half life of said proteinor peptide, it is understood that the albumin binder may contribute tothe overall increase of plasma half life.

In a preferred embodiment the linker moiety, if present, has from 2 to80 C-atoms, preferably from 5 to 70 C-atoms. In additional preferredembodiments, the linker moiety, if present, has from 4 to 20 heteroatoms, preferably from 2 to 40 hetero atoms, more preferably from 3 to30 hetero atoms. Particularly preferred examples of hetero atoms are N—,and O-atoms. H-atoms are not hetero atoms.

In another embodiment, the linker comprises at least one OEG molecule,and/or at least one glutamic acid residue, or rather the correspondingradicals (OEG designates 8-amino-3,6-dioxaoctanic acid, i.e. thisradical: —NH—(CH2)2-O—(CH2)2-O—CH2-CO—). In one preferred embodiment,the linker moiety comprises a di-carboxamide moiety and the linker islinked to a cysteine residue through a thioether bond. In preferredexamples, the di-carboxamide moiety contains from 2-30 C-atoms,preferably 4-20 C-atoms, more preferably 4-10 C-atoms.

In one preferred embodiment, the linker moiety comprises adi-carboxamide moiety linked to a sialic acid residue by an amide bond.In preferred examples, the di-carboxyl residue has from 2-30 C-atoms,preferably 4-20 C-atoms, more preferably 4-10 C-atoms. In additionalpreferred examples, the di-carboxyl residue has from 0-10 hetero-atoms,preferably 0-5 hetero-atoms.

In another preferred example, the linker moiety/spacer comprises a groupcontaining both an amino and a distal carboxyl-group linked to a sialicacid residue by an amide bond through its distal carboxyl groups. In onepreferred embodiment this group is an OEG group. The term “hydrophilicspacer” as used herein means a spacer that separates a monovalent DR3antibody/ligand according to the invention and an albumin bindingresidue with a chemical moiety which comprises at least 5 non-hydrogenatoms where 30-50% of these are either N or O. Preferably, the albuminbinding residue is, via a hydrophilic spacer, linked to a Cys residue.

The amino acid glutamic acid (Glu) comprises two carboxylic acid groups.Its gamma-carboxy group is preferably used for forming an amide bondwith an amino group of a sialic acid residue or a sialic acidderivative, or with an amino group of an OEG molecule, if present, orwith the amino group of another Glu residue, if present. The amino groupof Glu in turn forms an amide bond with the carboxy group of the halflife extending moiety, or with the carboxy group of an OEG molecule, ifpresent, or with the gamma-carboxy group of another Glu, if present.This way of inclusion of Glu is occasionally briefly referred to as“gamma-Glu”.

“Fc fusion derivatives” or “Fc fusion proteins” or DR3 antibody having amutated Fc domain is herein meant to encompass a DR3 ligand according tothe invention fused to an Fc domain that can be derived from anyantibody isotype, although an IgG Fc domain will often be preferred dueto the relatively long circulatory half life of IgG antibodies—IgG1 andIgG4 isotypes are preferred. The Fc domain may furthermore be modifiedin order to modulate certain effector functions such as e.g. complementbinding and/or binding to certain Fc receptors. The Fc domain mayfurthermore be modulated in order to increase affinity to the neonatalFc receptor. Fusion of a DR3 ligand according to the invention with anFc domain, having the capacity to bind to FcRn receptors, will generallyresult in a prolonged circulatory half life of the fusion protein.Mutations in positions 234, 235 and 237 in an IgG1 Fc domain willgenerally result in reduced binding to the FcγRI receptor and possiblyalso the FcγRIIa and the FcγRIII receptors. These mutations do not alterbinding to the FcRn receptor, which promotes a long circulatory halflife by an endocytic recycling pathway.

Preferably, a modified IgG1 Fc domain of a fusion protein according tothe invention comprises one or more of the following mutations that willresult in decreased affinity to certain Fc receptors (L234A, L235E, andG237A) and in reduced C1q-mediated complement fixation (A330S andP331S), respectively. Alternatively, the Fc domain may be an IgG4 Fcdomain optionally comprising the S241P/S228P mutation.

The term “antibody”, “monoclonal antibody” and “mAb” as used herein, isintended to refer to immunoglobulin molecules and fragments thereof thathave the ability to specifically bind to an antigen. Full-lengthantibodies comprise four polypeptide chains, two heavy (H) chains andtwo light (L) chains interconnected by disulfide bonds. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asHCVR or VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Antibodies can be in the form of different isotypes; e.g. IgG(e.g. IgG1, IgG2, IgG3, IgG4), IgGA1, IgA2, IgD, and IgE. A full-lengthantibody is normally bi-valent/di-valent, i.e. it has the capacity tobind to the antigen with both “arms”. In contrast, a mono-valentantibody according to the present invention comprises only one bindingsite specific for the antigen/DR3.

The “Fab region”/“Fab domain”/“Fab fragment”/“Fab”, contains variablesections that define the specific target that the antibody can bind. AFab fragment is an example of a mono-specific/mono-valent DR3 ligand/DR3antibody according to the present invention.

Examples of mono-valent DR3 ligands/antibodies according to the presentinvention include: Fab fragments, monovalent fragments consisting of theVL, VH, CL and CH I domains; a bivalent fragment comprising two Fabfragments linked e.g. by a disulfide bridge at the hinge region, whereonly one of these Fab fragments is specific for DR3; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;(vi) an isolated complementarity determining region (CDR); and (v) abi-specific antibody that is monovalent for DR3. Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426: andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Otherforms of single chain antibodies, such as diabodies are also encompassedwihin the term “monovalent DR3 ligands/antibodies”.

“Diabodies” are bivalent, bispecific antibodies in which VH and VLdomains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Hol-liger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

The terms “human antibody”, as used herein, means monovalent DR3antibodies according to the invention having variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular in the CDR3.

However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences, e.g. the so-called “humanized antibodies” orhuman/mouse chimeric antibodies. Humanized monovalent DR3 antibodies arealso a part of the present invention.

The term “chimeric monovalent antibody” refers to monovalent DR3antibodies according to the invention whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant region genes belonging to differentspecies. For example, the variable segments of genes from a mousemonoclonal antibody may be joined to human constant segments.

The term “epitope” as used herein means any antigenic determinant on anantigen to which the monovalent antibody binds. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar moieties and often have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Examples of methods for characterizing epitopes includeHX-MS, NMR, X-ray, peptide walking, assays, etc. The term “paratope”refers to the part of the antibody that recognizes the antigen.

Monovalent DR3 antibodies of the present invention may be described orspecified in terms of the epitope(s) or portion(s) of DR3 that theyrecognize or specifically bind. The epitope(s) or the polypeptideportions(s) may be specified as e.g. by N-terminal and C-terminalpositions, or by size in contiguous amino acid residues. Monovalent DR3antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide are included.

“Epitope binning”/“competition binding assay” refers to the use ofcompetitive binding assays to identify pairs of ligands/antibodies thatare, or are not, capable of binding DR3 simultaneously, therebyidentifying ligands/antibodies according to the invention that bind tothe same, or overlapping epitopes on the DR3 protein (se example 10), orthat cannot bind simultaneously due to steric hindrance. Binningexperiments provide evidence that antigenically distinct epitopes arepresent. However, by themselves, they do not identify, or “map” theepitope to a specific amino acid sequence or location on the DR3protein. Competition for binding can be evaluated for any pair ofligands/antibodies or fragments. Frequently, favourable properties of afamily (or bin) of ligands/antibodies can be correlated with a bindingto a specific epitope defined by the antibody bin/competitive group.

The terms “immunoreacts” or “immunoreacting”, as used herein, means anybinding of a ligand/antibody to its epitope with a dissociation constantKd lower than 10⁻⁴ M. The terms “immunoreacts” or “immunoreacting” areused where appropriate inter-changeably with the term “specificallybind”.

The term “affinity”, as used herein, means the strength of the bindingof a ligand/antibody according to the invention to an epitope. Theaffinity of an antibody/ligand is measured by the dissociation constantKd, defined as [Ab]×[Ag]/[Ab−Ag] where [Ab−Ag] is the molarconcentration of the antibody-antigen complex, [Ab] is the molarconcentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant Ka isdefined by 1/Kd. Preferred methods for determining antibody specificityand affinity by competitive inhibition can be found in Harlow, et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988), Colligan et al., eds., CurrentProtocols in Immunology, Greene Publishing Assoc. and WileyInterscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601(1983).

“Decreases IFN-gamma (IFN-γ) release in synovial fluid cells from RApatients” can be measured as described herein. It is understood that ata concentration of about 0.1, 0.5, 1, or 5 μg monovalent antibody/ml,using the assay conditions as described herein, IFN-γ release insynovial fluid T cells from RA patients is decreased by at least 15%,more preferably by at least 20%, more preferably by at least 25%, morepreferably by at least 30%, more preferably by at least 35%, morepreferably by at least 40%, more preferably by at least 45%, morepreferably by at least 50%, and most preferably by at least 60%, morepreferably by at least 70%, more preferably by at least 75%, morepreferably by at least 80%, and most preferably by at least 95%. Inresponding patient material, antibodies according to the inventiondecreases interferon release (e.g. IFN-gamma) in RA as well as CDpatient material.

“Decreases release of one or more cytokines in Lamina PropriaMononuclear Cells (LPMCs) from intestinal biopsies from CD patients” canbe measured as described herein. It is understood that at aconcentration of about 0.1, 0.5, 1, or 5 μg monovalent antibody/ml,using the assay conditions as described herein, cytokine release inLPMCs from CD patients is decreased by at least 15%, more preferably byat least 20%, more preferably by at least 25%, more preferably by atleast 30%, more preferably by at least 35%, more preferably by at least40%, more preferably by at least 45%, more preferably by at least 50%,and most preferably by at least 60%, more preferably by at least 70%,more preferably by at least 75%, more preferably by at least 80%, andmost preferably by at least 95%. In responding patient material,antibodies according to the invention decreases interferon release (e.g.IFN-gamma) in RA as well as CD patient material.

“Decreases release of one or more cytokines in CD4⁺ T cells” can bemeasured as described herein. It is understood that at a concentrationof about 0.1, 0.16, 0.5, 1, or 5 μg monovalent antibody/ml, using theassay conditions as described herein, cytokine release in CD4⁺ T cellsis decreased by at least 15%, more preferably by at least 20%, morepreferably by at least 25%, more preferably by at least 30%, morepreferably by at least 35%, more preferably by at least 40%, morepreferably by at least 45%, more preferably by at least 50%, and mostpreferably by at least 60%, more preferably by at least 70%, morepreferably by at least 75%, more preferably by at least 80%, and mostpreferably by at least 95%.

“Pharmaceutical compositions” comprising DR3 ligands according to theinvention may be supplied as a kit comprising a container that comprisesthe ligand according to the invention. Therapeutic polypeptides can beprovided in the form of an injectable solution for single or multipledoses, or as a sterile powder that will be reconstituted beforeinjection. Pharmaceutical compositions comprising ligands according tothe invention are suitable for subcutaneous and/or IV administration.Pharmaceutical compositions according to the present invention maycomprise one or more pharmaceutically acceptable carriers.

The term “treatment”, as used herein, refers to the medical therapy ofany human or other animal subject in need thereof. Said subject isexpected to have undergone physical examination by a medical orveterinary medical practitioner, who has given a tentative or definitivediagnosis which would indicate that the use of said specific treatmentis beneficial to the health of said human or other animal subject. Thetiming and purpose of said treatment may vary from one individual toanother, according to the status quo of the subject's health. Thus, saidtreatment may be prophylactic, palliative, symptomatic and/or curative.

In terms of the present invention, prophylactic, palliative, symptomaticand/or curative treatments may represent separate aspects of theinvention.

The ligands according to the present invention may be administered alongwith other drugs (e.g. methotrexate, dexamethasone, and prednisone)and/or other biological drugs.

The monovalent DR3 antibody/ligand may be produced by means ofrecombinant techniques. The DNA sequences encoding the monovalent DR3antibody/ligand according to the invention are usually inserted into arecombinant vector. The vector is preferably an expression vector inwhich the DNA sequence is operably linked to additional segmentsrequired for transcription of the DNA as well as a promoter capable ofdirecting the transcription of a cloned gene or cDNA in the desired hostcell.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically 1-2 days, to begin expressing the gene ofinterest. The host cell into which the DNA sequences encoding themonovalent DR3 antibody/ligand is introduced may be any cell, andincludes yeast, fungi, bacteria and higher eucaryotic cells. Examples ofmammalian cell lines for use in the present invention are COS-1, babyhamster kidney (BHK) and 293. A preferred BHK cell line is the tk-ts13BHK cell line that may be referred to as BHK 570 cells. In addition, anumber of other cell lines may be used within the present invention,including Rat Hep I, Rat Hep II, TCMK, NCTC 1469, CHO, and DUKX cells.

The transformed or transfected host cell described above is thencultured in a suitable nutrient medium under conditions permittingexpression of the monovalent DR3 antibody/ligand after which all or partof the resulting peptide may be recovered from the culture. Themonovalent DR3 antibody/ligand produced by the cells may then berecovered from the culture medium by conventional procedures includingseparating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. ionexchange chromatography, gelfiltration chromatography, affinitychromatography, or the like, dependent on the type of polypeptide inquestion.

Transgenic animal technology may be employed to produce the monovalentDR3 antibody/ligand of the invention. It is preferred to produce theproteins within the mammary glands of a host female mammal, preferablysheep, goats or cattle. Production in transgenic plants may also beemployed. Expression may be directed to a particular organ, such as atuber.

The monovalent DR3 antibody may also be obtained on basis of a bivalentantibody produced as described above and subsequently subject topeptidase digestion and isolation of the resulting Fab fragments.

Monovalent DR3 antibodies/ligands may subsequently beposttranslationally modified in order to obtain a protein having anextended in vivo circulatory half life.

LIST OF EMBODIMENTS

The following is a list of embodiments according to the invention. Thislist of embodiments is not intended to be limiting and it is understoodthat the present invention encompasses any combination of the followingembodiments.

Embodiment 1

A monovalent antagonistic DR3 antibody, wherein said monovalent antibodyblocks binding of DR3 to TL1A, and wherein said monovalent antibody in abivalent form thereof is an agonistic antibody that blocks binding ofDR3 to TL1A.

Embodiment 2

A monovalent antibody according to embodiment 1, wherein the monovalentantibody is not an antibody having the CDR sequences of the 11H08antibody set forth in WO2011106707 (SEQ ID NOs 14-15).

Embodiment 3

A monovalent antibody according to any one of embodiments 1-2, whereinsaid monovalent antibody is conjugated with a lipophilic moiety.

Embodiment 4

A monovalent antibody according to embodiment 3, wherein said lipophilicmoiety comprises a —(CH₂)_(n)—CO— fatty acyl group, wherein n is 16-18.

Embodiment 5

A monovalent antibody according to embodiment 3, wherein said lipophilicmoiety comprises a —(CH₂)_(n)—CO— fatty acyl group, wherein n is 15.

Embodiment 6

A monovalent antibody according to any one of embodiments 3-5, whereinsaid antibody is conjugated to a lipophilic moiety selected from thegroup consisting of formulas (I), (II), (III), (IV), (V), and (VI):

Embodiment 7

A monovalent antibody according to any one of embodiments 3-6, whereinsaid lipophilic moiety is attached to a naturally occurring cysteineresidue, preferably the C239 (Kabat numbering) cysteine residue, in theheavy chain of the antibody via a hydrophilic spacer.

Embodiment 8

A monovalent antibody according to any one of the preceding embodiments,wherein said antibody binds to an epitope on DR3, wherein said epitopecomprises I43 and/or L45 of SEQ ID NO 1.

Embodiment 9

A monovalent antibody according to any one of the preceding embodiments,wherein said antibody binds an epitope on DR3, wherein said epitopecomprises at least one of amino acids G37 to L45 and at least one ofamino acids L57 to A59 as set forth in SEQ ID NO 1.

Embodiment 10

A monovalent antibody according to embodiment 9, wherein said epitopecomprises amino acids G37 to L45 and amino acids L57 to A59 as set forthin SEQ ID NO 1.

Embodiment 11

A monovalent antibody according to any one of the preceding embodiments,wherein said ligand is a Fab fragment.

Embodiment 12

A monovalent antibody according to any one of the preceding embodiments,wherein said monovalent antibody binds DR3 with a dissociation constantof below 1 nM.

Embodiment 13

A monovalent antibody according to embodiment 12, wherein saidmonovalent antibody binds DR3 with a dissociation constant of below 500μM, preferably below 300 μM, preferably below 100 μM, most preferablypreferably below 30 μM.

Embodiment 14

A monovalent antibody according to any one of the preceding embodiments,wherein said antibody binds to the CRD1 domain of human DR3.

Embodiment 15

A monovalent antibody, alternatively a bivalent antibody, comprising thethree CDR sequences as set forth in SEQ ID NO 16 and the three CDRsequences as set forth in SEQ ID NO 17. In another embodiment, themonovalent antibody according to the invention comprises the three CDRsequences as set forth in SEQ ID NO 10 and the three CDR sequences asset forth in SEQ ID NO 11.

Embodiment 16

A monovalent antibody, alternatively a bivalent antibody, wherein saidantibody comprises a human frame work, the CDR3 sequence as set forth inSEQ ID NO 16 and the CDR3 sequence as set forth in SEQ ID NO 17 as wellas an “S49A” back mutation in the heavy chain.

Embodiment 17

A monovalent antibody according to embodiment 16, wherein said antibodycomprises the three CDR sequences as set forth in SEQ ID NO 16, and thethree CDR sequences as set forth in SEQ ID NO 17. In another embodiment,a monovalent antibody according to the invention comprises the heavychain and light chain as set forth in SEQ ID NO 16 and 17, respectively.

Embodiment 18

A monovalent antibody according to any one of embodiments 15-17, whereinsaid antibody is an IgG4 isotype.

Embodiment 19

A monovalent antibody according to any one of the preceding embodiments,wherein said antibody competes with monovalent antibody “0228” forbinding to human DR3, wherein the amino acid sequence of the 0228 heavychain is at set forth in SEQ ID NO 16 and the amino acid sequence of the0228 light chain is as set forth in SEQ ID NO 17. In another embodiment,the antibody binds to the same epitope as the 0228 antibody.

Embodiment 20

A monovalent antibody according to any one of embodiments 1-14, whereinsaid antibody comprises the three CDR sequences as set forth in SEQ IDNO 12 and the three CDR sequences as set forth in SEQ ID NO 13.

Embodiment 21

A monovalent antibody according to the invention, wherein said antibodycompetes with monovalent antibody 0124 for binding to human DR3, whereinthe amino acid sequence of the 0124 heavy chain is as set forth in SEQID NO 12 and the amino acid sequence of the light chain is as set forthin SEQ ID NO 13. In another embodiment, the antibody binds to the sameepitope as the 0124 antibody.

Embodiment 22

A monovalent antibody, alternatively a bivalent antibody, wherein saidantibody comprises the three CDR sequences as set forth in SEQ ID NO 18and the three CDR sequences as set forth in SEQ ID NO 19.

Embodiment 23

A monovalent antibody according to the invention, wherein said antibodycompetes with monovalent antibody 0130 for binding to human DR3, whereinthe amino acid sequence of the 0130 heavy chain is as set forth in SEQID NO 18 and the amino acid sequence of the 0130 light chain is as setforth in SEQ ID NO 19. In another embodiment, the antibody binds to thesame epitope as the 0130 antibody.

Embodiment 24

A monovalent antibody, alternatively a bivalent antibody, wherein saidantibody comprises the three CDR sequences as set forth in SEQ ID NO 20and the three CDR sequences as set forth in SEQ ID NO 21. (0143).

Embodiment 25

A monovalent antibody according to the invention, wherein said antibodycompetes with monovalent antibody 0143 for binding to human DR3, whereinthe amino acid sequence of the 0143 heavy chain is as set forth in SEQID NO 20 and the amino acid sequence of the 0143 light chain is as setforth in SEQ ID NO 21. In another embodiment, said antibody binds to thesame epitope as the 0143 antibody.

Embodiment 26

A monovalent antibody, alternatively a bivalent antibody, wherein saidantibody comprises the three CDR sequences as set forth in SEQ ID NO 22and the three CDR sequences as set forth in SEQ ID NO 23. (0152).

Embodiment 27

A monovalent antibody according to the invention, wherein said antibodycompetes with monovalent antibody 0152 for binding to human DR3, whereinthe amino acid sequence of the 0152 heavy chain is as set forth in SEQID NO 22 and the amino acid sequence of the 0152 light chain is as setforth in SEQ ID NO 23. In another embodiment, said antibody binds to thesame epitope as the 0152 antibody.

Embodiment 28

A monovalent antibody according to the invention, wherein said antibodydecreases IFN-gamma (IFN-γ) release in synovial fluid cells from RApatients, wherein said synovial fluid cells are co-stimulated with TL1A.Preferably, the cells are IL-12/IL-18-activated.

Embodiment 29

A monovalent antibody according to the invention, wherein said antibodydecreases release of one or more cytokines in Lamina Propria MononuclearCells (LPMCs) from intestinal biopsies from CD patients, wherein saidcytokines are selected from the list consisting of: IL-6, TNF-α, GM-CSF,and IFN-gamma (IFN-γ), and wherein said LPMCs are co-stimulated withTL1A, IL-12, and IL-18. Preferably, the cells are IL-12/IL-18-activated.

Embodiment 30

A monovalent antibody according to the invention, wherein said antibodydecreases release of one or more cytokines in CD4+ T cells, wherein saidcytokines are selected from the list consisting of: TNF-α, IL-6, GM-CSF,and IFN-gamma (IFN-γ), and wherein said T cells are co-stimulated byTL1A. Preferably, the cells are IL-12/IL-18-activated.

Embodiment 31

A monovalent antibody according to any one of the preceding embodiments,wherein the antibody is an IgG4 type antibody.

Embodiment 32

A monovalent antibody according to any one of the preceding embodiments,wherein said antibody is conjugated to one or more half life extendingmoiety selected from one or more of the list consisting of: fatty acidsand derivatives thereof, Hydroxy Ethyl Starch (HES), Poly Ethylen Glycol(PEG), hyaluronic acid (HA), heparosan polymers, Phosphorylcholine-basedpolymers, fleximers, dextran, poly-sialic acids (PSA), an Fc domain,transferrin, albumin, Elastin like peptides, XTEN polymers, albuminbinding peptides, and any combination thereof. It follows that themonovalent antibody according to the invention can be conjugated withtwo or more different types of half life extending moieties.

Embodiment 33

A monovalent antibody according to the invention, wherein saidmonovalent antibody comprises an Fc domain with reduced effectorfunctions or an Fc domain with increased stability. Preferably, the Fcdomain is an IgG1 Fc domain comprising one, two, three, four, or all ofthe following mutations: L234A, L235E, G237A, A330S, and P331S).Alternatively, the Fc domain may be an IgG4 Fc domain preferablycomprising the S241P/S228P mutation.

Embodiment 34

A monovalent antibody according to the invention, wherein saidmonovalent antibody is conjugated to a half life extending moiety via aglycan, preferably via a sialic acid.

Embodiment 35

A monovalent antibody according to the invention, wherein said antibodyis a human antibody.

Embodiment 36

A monovalent antibody according to the invention, wherein said antibodyis a humanized antibody.

Embodiment 37

A monovalent antibody according to the invention, wherein said antibodyblocks binding of one or more DR3 ligands. It is plausible that DR3binds to other ligands than TL1A. However, such ligands have not yetbeen identified.

Embodiment 38

A monovalent antibody (preferably comprising essentially the sameparatope as the 27F16A1 antibody) according to the invention, whereinsaid antibody comprises at least one of the CDR sequences in SEQ ID NO 8and at least one of the CDR sequences as set forth in SEQ ID NO 9.Preferably, said antibody comprises at two of the CDR sequences setforth in SEQ ID NO 8 and one of the CDR sequence set forth in SEQ ID NO9. More preferably, said antibody comprises three of the CDR sequencesas set forth in SEQ ID NO 8 and one of the CDR sequences as set forth inSEQ ID NO 9. More preferably, said antibody comprises at least one ofthe CDR sequences as set forth in SEQ ID NO 8 and two of the CDRsequences as set forth in SEQ ID NO 9. More preferably, said antibodycomprises at least one of the CDR sequences as set forth in SEQ ID NO 8and three CDR sequences as set forth in SEQ ID NO 9. More preferably,said antibody comprises one of the CDR sequences as set forth in SEQ IDNO 8 and one of the CDR sequences as set forth in SEQ ID NO 9. Morepreferably, said antibody comprises two of the CDR sequences as setforth in SEQ ID NO 8 and two of the CDR sequences as set forth in SEQ IDNO 9. More preferably, said antibody comprises three of the CDRsequences as set forth in SEQ ID NO 8 and three of the CDR sequences asset forth in SEQ ID NO 9. Any one of such antibodies according to thepresent invention may comprise one, two, three, four, five, or six ofsuch CDR sequences, wherein one or two amino acids from this or theseCDR sequences has been deleted, added, or mutated into a different aminoacid residue—thus resulting in one or more CDR sequences that aredifferent in one or more positions compared to the CDR sequences as setforth in SEQ ID NO 8 and SEQ ID NO 9.

Embodiment 39

A monovalent antibody according to the invention, wherein said antibodycompetes with monovalent antibody 27F16A1 for binding to human DR3,wherein the amino acid sequence of the 27F16A1 heavy chain is at setforth in SEQ ID NO 8 and the amino acid sequence of the 27F16A1 lightchain is as set forth in SEQ ID NO 9. In another embodiment, the presentinvention relates to antibodies binding to the same epitope as the27F16A1 antibody.

Embodiment 40

A monovalent antibody (preferably comprising essentially the sameparatope as the 27F44A2 antibody) according to the invention, whereinsaid antibody comprises at least one of the CDR sequences in SEQ ID NO10 and at least one of the CDR sequences as set forth in SEQ ID NO 11.Preferably, said antibody comprises at two of the CDR sequences setforth in SEQ ID NO 10 and one of the CDR sequence set forth in SEQ ID NO11. More preferably, said antibody comprises three of the CDR sequencesas set forth in SEQ ID NO 10 and one of the CDR sequences as set forthin SEQ ID NO 11. More preferably, said antibody comprises at least oneof the CDR sequences as set forth in SEQ ID NO 10 and two of the CDRsequences as set forth in SEQ ID NO 11. More preferably, said antibodycomprises at least one of the CDR sequences as set forth in SEQ ID NO 10and three CDR sequences as set forth in SEQ ID NO 11. More preferably,said antibody comprises one of the CDR sequences as set forth in SEQ IDNO 10 and one of the CDR sequences as set forth in SEQ ID NO 11. Morepreferably, said antibody comprises two of the CDR sequences as setforth in SEQ ID NO 10 and two of the CDR sequences as set forth in SEQID NO 11. More preferably, said antibody comprises three of the CDRsequences as set forth in SEQ ID NO 10 and three of the CDR sequences asset forth in SEQ ID NO 11. Any one of such antibodies according to thepresent invention may comprise one, two, three, four, five, or six ofsuch CDR sequences, wherein one or two amino acids from this or theseCDR sequences has been deleted, added, or mutated into a different aminoacid residue—thus resulting in one or more CDR sequences that aredifferent in one or more positions compared to the CDR sequences as setforth in SEQ ID NO 10 and SEQ ID NO 11.

Embodiment 41

A monovalent antibody according to the invention, wherein said antibodycompetes with monovalent antibody 27F44A2 for binding to human DR3,wherein the amino acid sequence of the 27F44A2 heavy chain is as setforth in SEQ ID NO 10 and the amino acid sequence of the light chain isas set forth in SEQ ID NO 11. In another embodiment, an antibodyaccording to the invention binds to the same epitope as the 27F44A2antibody.

Embodiment 42

A monovalent antibody (preferably comprising essentially the sameparatope as the 28F26A3 antibody) according to the invention, whereinsaid antibody comprises at least one of the CDR sequences in SEQ ID NO12 and at least one of the CDR sequences as set forth in SEQ ID NO 13.Preferably, said antibody comprises at two of the CDR sequences setforth in SEQ ID NO 12 and one of the CDR sequence set forth in SEQ ID NO13. More preferably, said antibody comprises three of the CDR sequencesas set forth in SEQ ID NO 12 and one of the CDR sequences as set forthin SEQ ID NO 13. More preferably, said antibody comprises at least oneof the CDR sequences as set forth in SEQ ID NO 12 and two of the CDRsequences as set forth in SEQ ID NO 13. More preferably, said antibodycomprises at least one of the CDR sequences as set forth in SEQ ID NO 12and three CDR sequences as set forth in SEQ ID NO 13. More preferably,said antibody comprises one of the CDR sequences as set forth in SEQ IDNO 12 and one of the CDR sequences as set forth in SEQ ID NO 13. Morepreferably, said antibody comprises two of the CDR sequences as setforth in SEQ ID NO 12 and two of the CDR sequences as set forth in SEQID NO 13. More preferably, said antibody comprises three of the CDRsequences as set forth in SEQ ID NO 12 and three of the CDR sequences asset forth in SEQ ID NO 13. Any one of such antibodies according to thepresent invention may comprise one, two, three, four, five, or six ofsuch CDR sequences, wherein one or two amino acids from this or theseCDR sequences—thus resulting in one or more CDR sequences that aredifferent in one or more positions compared to the CDR sequences as setforth in SEQ ID NO 12 and SEQ ID NO 13.

Embodiment 43

A monovalent antibody according to the invention, wherein said antibodycompetes with monovalent antibody 28F26A3 for binding to human DR3,wherein the amino acid sequence of the 28F26A3 heavy chain is as setforth in SEQ ID NO 12 and the amino acid sequence of the 28F26A3 lightchain is as set forth in SEQ ID NO 13. In another embodiment, theantibody binds to the same epitope as the 28F26A3 antibody.

Embodiment 44

A DNA molecule encoding a monovalent antibody or ligand according to theinvention.

Embodiment 45

An expression vector comprising a DNA molecule according to embodiment44.

Embodiment 46

A host cell comprising an expression vector according to embodiment 45,or a DNA molecule according to embodiment 44.

Embodiment 47

A method for making a ligand or an antibody according to the invention,wherein said method comprises incubation of a host cell according toembodiment 46.

Embodiment 48

A pharmaceutical composition comprising a compound according to theinvention. The composition optionally comprises at least onepharmaceutically acceptable carrier/excipient.

Embodiment 49

Use of a ligand according to the invention or a pharmaceuticalcomposition according to the invention as a medicament.

Embodiment 50

Use of a ligand according to the invention, or a pharmaceuticalcomposition according to the invention for treating an inflammatorydisease.

Embodiment 51

Use of a ligand according to the invention, or a pharmaceuticalcomposition according to the invention, for treatment of RA.

Embodiment 52

Use of a ligand according to the invention, or a pharmaceuticalcomposition according to the invention, for treatment of Crohns Disease(CD).

Embodiment 53

Use of a ligand according to the invention, or a pharmaceuticalcomposition according to the invention, for treatment of ulcerativecolitis (UC).

Embodiment 54

A method of treating an inflammatory disease, wherein said methodcomprises administering a ligand according to the invention, or apharmaceutical composition according to the invetnion, to a person inneed thereof. The inflammatory disease is preferably RA, Crohns diseaseor ulcerative colitis.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

EXAMPLES Example 1 Immunization and Hybridoma Generation

As stated in the following examples it is difficult to produce a solubleform of full length DR3 of high quality due to the cysteine rich natureof this protein. Soluble forms of DR3 tend to be highly aggregated,which can lead to shielding of important ligand-binding regions and makeit difficult to raise desired antibody responses upon immunization. Awhole range of immunization-regimens have been performed in order toobtain a robust anti-DR3 antibody (Ab) serum titer using both solublefull-length DR3 protein, DR3 protein-derivatives containing only partsof the ECD (like e.g. immunization with only the CRD1 domain) of DR3,and DR3-expressing cells (as described in example 3) for immunization.Immunizations have been performed in different mice strains (BALB/C, RBFand NMRCF1) in order to increase the antibody-repertoire diversity andthe likelihood of generating neutralizing anti-DR3Abs. In one example,BALB/C mice were immunized eight times intraperitoneally (IP) with 5×10⁶DR3-expressing CHO cells with or without incomplete Freunds adjuvant(IFA), followed by 6 IP immunizations with purified hDR3-Fc (see example3) in IFA. Mice sera were screened for DR3/hTL1A blocking antibodies byFACS as described in Example 4. Mice having a DR3/hTL1A blocking Abtiter received a final boost consisting of a single intravenousinjection (i.v.) in the tail vein using DR3-mFc. Three days after boostthe mice were sacrificed and splenocytes were fused with X63Ag8653myeloma cells by standard electrofusion procedure. The fused cells wereseeded in 96-well plates and cultured in DMEM (Invitrogen) and HATselection medium supplemented with FCS (Hyclone) for one week prior toscreening for DR3/hTL1A blocking mAbs.

Example 2 Cells for Screening and Immunization

Cells over-expressing transmembrane (TM) DR3 have been developed as anessential tool for use in cell based screening assays and forimmunisation of mice.

Production of a stable cell line overexpressing TM DR3 has not beenstraight-forward. Initially full length DR3 including TM and deathdomain (DD) (see SEQ ID NO 1 for full AA sequence) was cloned into thepcDNA3.1 expression vector (Invitrogen) and the vector was used fortransfection of Ba/F3 cells. However, the DD in full length DR3 leads tocell death in stable cell lines. It was simply not possible to expressfull length DR3 with an active DD. In order to inactivate the DD in DR3and still be capable of expressing transmembrane DR3 protein two newconstructs were designed and developed. A mutation in SEQ ID NO 1(L356N) according to Itoh and Nagata, JBC (268) pp. 10932-10937, 1993who described a similar mutated version for CD95 where the mutationleads to inactivation of the receptor. Deletion of the DD in SEQ ID NO 1(ΔM339-P417) as determined according to Screaton et al., PNAS (94) pp.4615-4619, 1997. Both the truncated version and the mutated version wascloned into pcDNA3.1 and expressed in Ba/F3. This resulted in two stablecell lines overexpressing surface DR3. From flow-cytometry (FCM)analysis the expression level for DR3 was found to be highest in thecell line developed with the truncated version of DR3. However, theexpression level was still too low for a cell line to be used inscreening assay. It was therefore decided to investigate whether theGS-CHO system from Lonza (Basel, Switzerland) could be used forexpression of the truncated version of DR3. The coding cDNA frompcDNA3.1 was transferred to the Lonza expression vector, pEE14.4.

The establishment of stable CHOK1SV cells were performed according toLonzas manufacturer's protocol. CHOK1SV cells were transfected byelectroporation. Prior to transfection the pEE14.4 plasmid waslinearized by digesting with the AcII restriction enzyme. Ten μg of AcIIdigested pEE14.4 plasmid was used for transfection of 1×10⁷ CHOK1SVcells by electroporation. Cells were plated into a T75 flask. The dayafter the transfection selection was initiated by adding L-Methioninesulfoximine (MSX) to a final concentration of 50 μM in CD-CHO mediumwithout glutamax (Invitrogen). Three weeks after transfection, the cellsare purified by using lympholyte-mammal (Cedarlane), transferred toshakerflask and cultured in an incubator shaker at 37° C., 8% CO₂ and125 rpm. The selection pressure was from this point lowered to 25 μMMSX. DR3 expression was analysed by FCM. However the DR3 expressionlevel was very low and it was decided to sort the pool byfluorescence-activated cell sorting (FACS) to increase the expressionlevel. Four days after FACS the pool were tested for DR3 expression byFCM and for the first time DR3 expression was fine (FIG. 2).Unfortunately, after 2-3 weeks the DR3 expression level starts todecrease and the cells could no longer bee used for screening.

In an attempt to increase the DR3 level and make the pool more stablethe MSX selection pressure was investigated by increasing to 50 μM and100 μM and in addition the pool was subcloned with ½ cell/well in 4×96Wplates. Subcloning resulted in two clones with high DR3 expression levelanalysed by FCM. However after 1-3 weeks the clones start losing theexpression again and appear unstable and thus could not be used forscreening. Increasing the MSX selection to 100 μM for the pool resultedin a very fine and high expression level and in addition this could keepa high expression level over time (FIG. 3). The resulting cell line usedfor screening and immunisation are therefore a CHOK1SV transfected withtruncated DR3 and cultured in CD-CHO+100 μM MSX.

Example 3 Expression and Purification of Soluble DR3

Human DR3 is a very cysteine rich protein comprising four cysteine-richdomains (CRD1, CRD2, CRD3 and CRD4) in the extracellular domain (ECD,residues 25-199) and a “death domain” (DD) in the cytoplasmic domain.According to Banner et al., Cell (73) pp. 431-445, (1993) each CRDtypically contains six cysteine residues that form three disulfidebounds. In addition each CRD can be subdivided into modules A1 and B2which are typically observed in conventional members of the TNFRsuperfamily (J. H. Naismith et al., Trends Biochem. Sci. (23) pp. 74-79,1998). The sequence for DR3 with predicted CRDs and modules A1 and B2 isshown in FIG. 4.

Production of soluble DR3 has been very difficult, which may be due tothe high cysteine content of the protein. Recombinant expression of theECD of DR3 in human cell lines usually led to the secretion of solubleproteins that contained large amounts of oligomers and high molecularweight complexes, due to the formation of intermolecular disulfidebonds. These protein batches did not bind TL1A and were not active incellular assays.

Different approaches have been taken to solve the aggregation problems.Initially, the full ECD (25-199) of DR3 was fused to a Fc domain ofeither mouse IgG1 or human IgG4. Transient production in HEK293 resultedin low yields (<10 mg/L) and a high degree of aggregation. Gelfiltration allowed enrichment of the expected dimer, but due to the lowyields and high degree of oligomerization, it was initially not possibleto obtain a pure fraction. Other purification tags such asbiotinylation, FLAG-tag or the trimerization peptide, Tenascin C (TNC)have also been tested, but did not improve results.

In addition, DR3-Fc fusion proteins have been engineered that containedonly parts of the ECD of DR3, in order to determine the part of theprotein responsible for the oligomerisation. Four proteins were designedas follows:

DR3 (CRD1)-Fc (SEQ ID NO 2) DR3 (CRD1+A1)-Fc (SEQ ID NO 3) DR3(CRD1+CRD2)-Fc (SEQ ID NO 4) DR3 (CRD1+CRD2+A1)-Fc (SEQ ID NO 5)

The formation of intermolecular disulfide bonds and thereby misfoldingcorrelated with increasing length of the DR3 ECD, probably reflectingthe increasing number of cysteines. Concurrently, the yield of proteinexpression decreased with increasing length of the DR3 EC domain. Gelfiltration allowed the purification of mainly dimeric fractions for allthese constructs; however, the proteins were not biologically active.Apart from the constructs a number of truncation and cys-deletionconstructs were also made in the attempt to pinpoint a single cysteineor a minor region responsible for the oligomerization. However, nosingle region or specific cysteine seem to be responsible and the entireDR3 sequence seems to be difficult to express in a soluble form.

In addition, a fusion protein between the glutathion S-transferase (GST)and the CRD1 of DR3 has been produced, that was homogenic (SEQ ID NO 6).

Stable expression of the DR3-Fc, comprising the ECD of DR3, a linkercontaining a TEV cleavage site and the Fc part of human IgG4, in ChineseHamster Ovary (CHO) cells increased the expression levels and allowedfor the isolation of mainly dimeric protein (SEQ ID NO 7). This fractioncould compete for TL1A binding to DR3 in a reporter gene assay measuringTL1A induced DR3 signalling.

A number of these proteins have been used for immunization of mice. Someof the batches were further purified to separate out small amountsnon-oligomerized DR3. These batches were used for surface plasmonresonance (SPR) binding assays.

Transient Expression in HEK293 Cells: Vectors:

All the variants of the DR3 proteins were expressed by using the CMVpromotor-based expression vectors (pTT vectors). The pTT vectors aregenerated for transient expression in the HEK293-6E EBNA-basedexpression system developed by Yves Durocher (Durocher et al. NucleicAcid Research, 2002).

Cell Maintenance:

HEK293-6E cells were grown in suspension in FreeStyle™ 293 expressionmedium (Gibco) supplemented with 25 μg/ml Geneticin (Gibco), 1% v/v ofthe surfactant Pluronic F-68 (Gibco) & 1% v/v Penicillin-Streptomycin(Gibco). Cells were cultured in Erlenmeyer shaker flasks in shakerincubators at 37° C., 8% CO₂ and 125 rpm and maintained at celldensities between 0.1-1.5×10⁶ cells/ml.

DNA Transfection:

The cell density of cultures used for transfection was 0.9-1.1×10⁶cells/ml.

One μg DNA was used per ml cell culture.

The DNA was diluted in Opti-MEM media (Gibco) 30 μl media/μg DNA, mixedand incubated at room temperature (23-25° C.) for 5 min.

293Fectin™ (Invitrogen) was used as transfection reagent at aconcentration of 1 μl per μg DNA.

The 293Fectin™ was diluted 30× in Opti-MEM media (Gibco), mixed andincubated at room temperature (23-25° C.) for 5 min.

The DNA and 293Fectin solutions were mixed and left to incubate at roomtemperature (23-25° C.) for 25 min.

The DNA-293Fectin mix was then added directly to the cell culture.

The transfected cell culture was transferred to a shaker incubator at37° C., 8% CO₂ and 125 rpm.

5-6 days post transfection, cell culture supernatants were harvested bycentrifugation, followed by filtration through a 0.22 μm PES filter(Corning).

When possible quantitative analysis of protein production was performedby Biolayer Interferometry using the ForteBio Octet system and protein Abiosensors.

Purification of DR3-Fc proteins

Cell supernatants were loaded directly onto a column with Protein Aaffinity beads (MabSelect Sure; GE Healthcare) with 20 mM Tris/HCl, pH8.5 as buffer. Bound proteins were eluted stepwise with 10 mM, 50 mM and100 mM sodium formiate pH 3.5. Eluted fractions were concentrated andshifted to PBS buffer by ultrafiltration. Gel filtration was carried outon a Superdex200 column (GE Healthcare) with PBS as buffer. Fractionscontaining dimeric proteins were pooled and concentrated byultrafiltration. All chromatography steps were performed with an AktaExplorer FPLC instrument (GE Healthcare).

Example 4 Screening for Antibodies Blocking the TL1A:DR3 InteractionPre-Screening of Mice Sera

It turned out essential to pre-screen the mice sera for blocking effectbefore performing fusions, since the mice had a very high difference indegree of blocking effect in sera.

Mice have been immunized with both DR3 over-expressing cell-lines andDR3 protein. A whole range of immunization-regimens have been performedfor obtaining different mice with different antibody-repertoires. Wholecell immunization includes injection of multiple antigens in contrast tosingle antigen immunization. It has previously been shown that moreimmunization optimization is needed when using whole cell immunization,as different immunizations-regimens give rise to different antibodyrepertoires. Screenings mice sera for blocking effect have been animportant tool in order to select mice for fusions (FIG. 5).

Screening of Hybridoma Supernatants

In order to select for antibodies against DR3 with the ability to blockTL1A binding, the screening plan has been as follows:

As a primary test for DR3 specific antibodies, a high-throughputimage-based assay was set up. This was done in an FMAT system, byscreening for the ability to bind DR3 transfected CHO cells andcounter-screened against wild-type cells (see example 2 for generationof the DR3 cell line).

Assay was done by labelling 50 μl of cell-preparations (50000 cells prwell in 50 μl) to 96 well plates (Greiner, cat no. 65021) together with25 μl of diluted monoclonal antibodies in titration or hybridomasupernatant undiluted and incubated at 4° C. for 1 hour. Afterincubation and wash (PBS buffer with 5% Fetal calf serum, centrifuge for5 minutes at 200 g) 50 μl of secondary antibody pr well (diluted in PBSbuffer 1:400) was added and incubated for another 1 hour at 4° C. APCanti mouse were used when screening from RBF mice or Balb/c miceJacksons #115-136-071 1:400.

Finally cells were washed and transferred to black FMAT plates were readon a PerkinElmer Envision Turbo (FMAT) measuring fluorescence.

The next part of the initial screening was to test for the ability toblock the DR3 ligand (TL1A) binding. This was done on the selected DR3specific hybridoma supernatants, by screening the positivehybridoma-supernatant for the ability to block the TL1A-flag tag bindingto DR3 transfected cells. As a control non-transfected cells irrelevantantibodies or supernantants were used.

FCM based inhibitions assay was done by adding 50 μl of dilutedmonoclonal antibodies in titration or hybridoma supernatant undiluted tothe DR3 positive cells (50000 cells pr well) and incubated at 4° C. for1 hour. After incubation and wash (PBS buffer with 5% Fetal calf serum,centrifuge for 5 minutes at 200 g) TL1A-flag tag (conc. In total volumeof 50 μl) where added to 96 well plates (Greiner, cat no. 65021). Againafter incubation and wash secondary antibody (APC anti FLAG tag ab fromPerkin Elmer in a Final conc. of 5 μg/ml) (diluted in PBS buffer 1:400)was added and incubated for another 1 hour at 4° C.

In parallel a reporter gene assay as described in example 5 whereperformed in order to detect for agonistic or antagonistic functionalityof the antibodies.

In house produced TL1A is the mature extracellular domain of TL1A. TheTL1A contains an N-terminal FLAG-HIS-TEV tag. FLAG-tag is used fordetection in FACS/FMAT, HIS-tag is used for purification and the TEVsite allows for optional TEV cleavage and removal of the tag. TL1Aproduced in E. coli is commercially available from R&D Systems. In houseTL1A was produced in HEK cells and contained two N->Q point mutations inorder to disrupt the potential N-glycosylation sites. Recombinantcynomolgus TL1A was produced similarly.

Example 5 DR3 Reporter Gene Assay

It was reported by Chinnaiyan et al. 1996 (Science yr:1996 vol:274iss:5289 pg:990-992) that DR3 signals via NFkappaB and induces apoptosiswhen DR3 are ectopicly expressed in MCF7 cells. We wanted to utilize theNFkappaB signalling as a reporter and to use a reduced DR3 expression toavoid apoptotic cells.

The HEK293 cell line was stably transfected with a human DR3 expressionplasmid and a NFkappaB-luciferase reporter plasmid. The DR3 expressionplasmid is based on pcDNA3.1 (hygro)+ and contains a hygromycinresistance gene regulated by the SV40 promoter, the CMV promoter wasremoved and two heat-shock-elements (HSE), a minimal c-fos promoter andhuman DR3 coding sequence were inserted. The reporter is based onpGL3-basic and the Luc+ response is regulated by three NFkappaB sitesand a minimal interferon promoter. The neomycin resistance generegulated by the SV40 promoter was inserted in the vector.

A DR3/HEK293 reporter gene cell, named 15, gives a 20-25 fold responsein luciferase activity on 40 ng/ml TL1A (R&D Systems) stimulation. Theprocedure to detect the effect of TL1A, agonizing antibodies andantagonizing antibodies/Fab are as follows. The 15 cells are seeded20.000 cells/well in polyD-lysine coated black view 96 well plates. Thefollowing day TL1A or medium are added to the wells and after 4 hours ofincubation the Steady-GLO kit (Promega) is used and luminescence aremonitored by a TopCount NXT (Perkin Elmer) instrument. In order todetermine a neutralizing effect, antibodies or Fabs are preincubatedwith cells for 15 minutes before addition of TL1A.

For screening of hybridoma supernatants, all samples were tested in atleast duplicates and using both 0 ng/ml, 10 ng/ml TL1A and 50 ng/mlTL1A.

Example 6 Antibodies and Fabs Blocking the TL1A:DR3 Interaction

Monoclonal antibodies (mAb) with specificity for DR3 and tested positivefor blocking of TL1A interaction with DR3 were identified. A total ofseveral hundreds specific DR3 mAb have been selected after at least 50fusions from mice immunized with primarily cells expressing DR3. Furthertesting of the DR3 specific clones for TL1A inhibition/blocking gave 74clones. FIG. 6 shows some of the positive antibodies blocking DR3antibodies. Also included are 2 positive DR3 specific clones withoutsuch blocking effect.

All of the mAbs were selected for further studies along with Fab versionof the mAbs. Table 1 summarises a study performed and provide thecorrelation between mAb and Fab numbering and the hydridoma clonenumbering.

TABLE 1 List of mAbs and Fabs selected for further studies andcorrelation between mAb and Fab numbering and the hydridoma clonenumbering. Block TL1A TL1A-induced CD4+ sequence Reporter to DR3 T-cellproliferation Prepared clone (see gene assay binding assay CompoundFormat from name example 9) (see example 7) (see example 6) (see example8) 0070 mAb hybridoma 27F16A1 + agonistic yes n.d. supernatant 0071 mAbhybridoma 27F38 n.d. weak no n.d. supernatant agonistic 0072 mAbhybridoma 27F44A2 + agonistic yes see example 8 supernatant 0073 mAbhybridoma 28F26A3 + agonistic yes see example 8 supernatant 0074 mAbhybridoma 28F69 n.d. weak no n.d. supernatant agonistic 0075 mAbhybridoma 29F6 n.d. weak no n.d. supernatant agonistic 0076 mAbhybridoma 29F8 n.d. agonistic yes n.d. supernatant 0077 mAb hybridoma30F1 n.d. weak no see example 8 supernatant agonistic 0087 Fab papainn.a. + antagonistic yes see example 8 cleavage of mAb 0072 0088 Fabpapain n.a. + antagonistic yes see example 8 cleavage of mAb 0073 0089Fab papain n.a. n.d. antagonistic yes n.d. cleavage of mAb 0076 0091 Fabpapain n.a. n.d. no effect no see example 8 cleavage of mAb 0077 n.a.:not applicable n.d.: not determined

Antibodies 0072, 0073 and 0076 were found to block the TL1A:DR3interaction in a dose-dependent manner. The corresponding Fab molecules(0087, 0088 and 0089) retained this ability also in a dose-dependentmanner (FIG. 7). Correspondingly, a Fab derived from a DR3 binding mAbthat was not able to inhibit the TL1A:DR3 interaction, also did not havethis ability (compounds 0077 and 0091, FIG. 7).

Table 2 show examples of Fab characteristics: In total we characterized46 Fabs for binding, ability to block TL1A-DR3 interaction, TL1A inducedT cell activation, binding (more details in other examples). A number ofselected Fab's together with controls are shown in the table:

TABLE 2 Fab characteristics Fabs tested for displacement/blocking ofTL1A binding to hDR3 expressing CHO cells Blocking/displacement of TL1AInhibition assay binding shown as IC50 value (nM) Format of Fab and asmax percentage inhibition. Fab ID n: IC50 nM % inhibition 00228IgG4-Cys, hz 1 0.484 78 00123 IgG4-Ser, chimeric 8 0.443 80 00124IgG4-Ser, chimeric 4 1.297 79 00130 murine Fab 2 1.340 73 00143IgG4-Ser, chimeric 2 0.120 76 00152 IgG4-Ser, chimeric 1 0.512 53 00231IgG4-Ser, hz 1 24.680 81 00158 IgG4-Ser, hz 11 No inhib 10 00148IgG4-Ser, chimeric 2 9.221 33 00163 IgG4-Ser, chimeric 2 No inhib 1200122 IgG4-Ser, chimeric 2 5.114 33 n: number of determinations, Noinhib: No inhibition seen.

Example 7 Functional Effect of DR3 Antibodies and Derived Fabs

Monoclonal antibodies (mAb) raised against DR3 and tested positive forblockade of TL1A interaction with DR3 were potential neutralizers of DR3signalling.

The human DR3 reporter gene cell line was incubated with the antibodiesfor 15 minutes followed by either stimulation with TL1A or addition ofmedium. The antibodies did only at very high concentrations have amodest neutralization of TL1A action, by contrast they were efficientactivators themselves at lower doses.

Table 3 shows the effect of the 0072 antibody. The activation profile of0072 without addition of TL1A is bell-shaped, indicating that thebivalent antibody activates the DR3 by cross-binding the receptor. Theactivation increases until a certain concentration whereupon itdecreases DR3 activation including the TL1A mediated activation. Athigher concentration of antibodies all the paratopes are not any longeroccupied by receptor but the antibody is still blocking for TL1A bindingand activation and an inhibitory effect is seen. The TL1A specificantibody MAB7441 (R&D Systems) is included to emphasize that theactivation of DR3 is TL1A specific.

TABLE 3 The 0227-0000-0072 anti-DR3 antibody tested on human DR3reporter cell line 2.5 0.000256 0.00128 0.0064 0.032 0.08 4 20 100microg/ml microg/ microg/ microg/ microg/ microg/ microg/ microg/microg/ Medium MAb7441 ml-0072 ml-0072 ml-0072 ml-0072 ml-0072 ml-0072ml-0072 ml-0072 Fold 1 1.1 1.1 2.2 9.3 18.6 25.1 21.1 16.3 9.2 responseTL1A + TL1A + TL1A + TL1A + TL1A + TL1A + TL1A + TL1A + TL1A + 40 2.50.000256 0.00128 0.0064 0.032 0.08 4 20 100 ng/ml microg/ml microg/microg/ microg/ microg/ microg/ microg/ microg/ microg/ TL1A MAb7441ml-0072 ml-0072 ml-0072 ml-0072 ml-0072 ml-0072 ml-0072 ml-0072 Fold32.8 3.7 33.8 37.2 40.3 41.0 38.4 34.0 27.8 18.4 response

Based on these data we hypothesized that a monovalent antibody withaffinity to DR3, e.g. a Fab (fragment ab), would probably not clusterthe receptors but might still inhibit TL1A binding. The antibodies werecleaved with papain and Fab fragments were purified. The ability of Fabfragments to inhibit TL1A activation of DR3 was tested in the human DR3reporter assay. Fab fragments from four different DR3 antibodies weretested and the three (0072, 0073 and 0076 corresponding to Fab 0087,0088, 0089) of them could block TL1A activation (Table 4).

TABLE 4 Fabs derived from anti-DR3 antibodies tested on human DR3reporter cell line 2.5 0.1 microg/ml 1 microg/ml 10 microg/ml 100microg/ml microg/ml anti-DR3 anti-DR3 anti-DR3 anti-DR3 Medium MAb7441molecule molecule molecule molecule Fold 1.0 1.0 1.0 1.0 1.0 1.0response- 0087 Fab Fold 1.0 1.0 1.1 1.1 1.1 1.0 response- 0088 Fab Fold1.0 1.0 1.1 1.1 1.1 1.1 response- 0089 Fab Fold 1.0 1.0 1.1 1.0 1.1 1.1response- 0097 Fab TL1A + TL1A + 0.1 TL1A + 1 TL1A + 10 TL1A + 100 2.5microg/ml microg/ml microg/ml microg/ml 40 ng/ml microg/ml anti-DR3anti-DR3 anti-DR3 anti-DR3 TL1A MAb7441 molecule molecule moleculemolecule Fold 24.0 2.3 5.8 2.7 2.1 2.1 response- 0087 Fab Fold 22.6 2.015.2 6.3 2.9 2.8 response- 0088 Fab Fold 22.5 1.8 17.1 11.2 7.6 7.5response- 0089 Fab Fold 25.6 2.3 26.7 25.7 24.9 22.0 response- 0097 Fab

The fourth Fab 0097 (from mAb 0077) did not inhibit and this is inaccordance with mAb 0077 not blocking TL1A binding to DR3 (FIGS. 6 and7). Table 5 show the ability of the four mAbs (-0072, -0073, -0076 and-0077) to activate the DR3 gene reporter assay in absence of TL1A, alsohere -0077 prove to be a very poor agonist.

TABLE 5 Anti-DR3 antibodies tested on human DR3 reporter cell line 0.1 110 100 2.5 microg/ml microg/ml microg/ml microg/ml microg/ml anti-DR3anti-DR3 anti-DR3 anti-DR3 Medium MAb7441 molecule molecule moleculemolecule Fold 1.0 1.0 20.6 19.8 17.3 8.5 response- 0072 antibody Fold1.0 1.0 12.8 14.4 12.8 6.9 response- 0073 antibody Fold 1.0 1.0 18.322.7 17.5 9.0 response- 0076 antibody Fold 1.0 1.0 1.1 1.2 2.8 10.3response- 0077 antibody TL1A + 0.1 TL1A + 1 TL1A + 10 TL1A + 100 TL1A +2.5 microg/ml microg/ml microg/ml microg/ml 40 ng/ml microg/ml anti-DR3anti-DR3 anti-DR3 anti-DR3 TL1A MAb7441 molecule molecule moleculemolecule Fold 24.0 2.3 29.6 24.8 23.3 13.6 response- 0072 antibody Fold22.6 2.0 24.2 27.9 25.5 19.0 response- 0073 antibody Fold 22.5 1.8 27.527.8 25.6 19.5 response- 0076 antibody Fold 25.6 2.3 23.7 23.3 25.1 24.9response- 0077 antibody

Recombinant Fabs were tested in the “I5” human DR3 reporter cell lineassay and IC50 was determined when 40 microg/ml TL1A was used, see table6. Two versions of the 11H08 anti-DR3-Fabs, H1L2 and H1L4, from thepatent WO2011/106707, were also included. The H1L2 (0227-0000-0230) andH1L4 (0227-0000-0231) has an IC50 of 3.7 nM and 2.8 nM, respectively,whereas 0227-0000-0228 is ten times lower, 320 μM.

TABLE 6 Fab's tested on the human DR3 reporter gene cell line (I5)Derived from IC50 at 40 ng/ml Fab ID antibody TL1A [Molar]0227-0000-0123 27F44 188 pM 0227-0000-0228 27F44 320 pM 0227-0000-0230H1L2 (11H08) 3.7 nM (WO2011106707) 0227-0000-0231 H1L4 (11H08) 2.8 nM(WO2011106707) 0227-0000-0124 28F26 2.4 nM 0227-0000-0130 45F36 1.4 nM0227-0000-0143 44F434 290 pM 0227-0000-0152 50F191 2.1 nM 0227-0000-0158Hz-aTNP Isotype control 0227-0000-0148 5F13 Not antagonistic

The cynomolgus DR3 reporter cell line was generated similar to the humanreporter cell line. The stable cynomolgus DR3 reporter cell line wasnamed Lyda20 and gives a 10-14-fold response by adding 40 ng/ml human(0227-0000-0011) or cynomogus (0227-0000-0141) soluble TL1A. In Table 7,data for 0123 and 0228 activation of cynomolgus DR3 is show.

TABLE 7 Fabs tested on the cynomolgus DR3 reporter gene cell line(Lyda20) Fab ID IC50 at 40 ng/ml TL1A 0227-0000-0123 236 nM0227-0000-0228 628 nM

The marmoset DR3 reporter cell assay is a transient assay, theNFkappaB-reporter plasmid and marmoset DR3 expression vector regulatedby two heat-shock-element and a minimal cFos promoter were transfectedinto HEK293 cells using FuGene 6 transfection reagent (Roche) two daysbefore stimulation with TL1A. In table 8, activation of DR3 from threespecies is shown for a selection of Fabs.

TABLE 8 Fabs tested on the transiently transfected primate DR3 reportercells IC50 of at IC50 of at IC50 of at 40 ng/ml 40 ng/ml TL1A, 40 ng/mlTL1A, human cynomolgus TL1A, marmoset DR3 expressed DR3 expressed DR3expressed Fab ID HEK293 HEK293 HEK293 0227-0000-0123 330 pM 99 nM 323 pM0227-0000-0228 355 pM 73 nM 549 pM 0227-0000-0230 4.2 nM 14 nM 219 nM0227-0000-0231 4.3 nM 13 nM 377 nM

Fab molecules derived from the 27F44 antibody have a very poor IC50 whentested in the transient cynomolgus DR3 reporter cells, as shown in table8, 99 and 73 nM. However, the transient marmoset DR3 reporter cell assayshowed an IC50 value similar to the transient human DR3 assay.

The 11H08/H1L2 (0227-0000-0230) and 11H08/H1L4 (0227-0000-0231) Fabs hasan IC50 of 4.2 and 4.3 nM in the transient human DR3 reporter assay,three times higher IC50 on the transient cynomolgus DR3 reporter assay,14 and 13 nM, and finally 50 times higher IC50 in the transient marmosetDR3 reporter gene assay, 219 and 377 nM. All numbers are listed in table8.

Example 8 Binding to Activated Human Cells by Flow Cytometry

Buffy Coats were obtained from normal healthy volunteers from CopenhagenUniversity Hospital. CD4+ T cells were isolated through magnetic beadseparation. Cells were activated with 2 ng/ml IL-12, 50 ng/ml IL-18 and100 ng/ml TL1A (Flag-HIS-TEV-TL1A-produced at Novo Nordisk A/S) andcultured for 5 days. On day 5 cells were stained with 10, 5, 1, 0.5, 0.1or 0.0001 μg/ml of the anti-DR3 mAb clones 0072, 0073, or 0077, or withFab fragment clones 0087, 0088 and 0091. A secondary PE conjugatedgoat-anti-mouse (H+L) was used for detection.

Results: The monoclonal IgG clone 0077, and its corresponding Fab clone0091 demonstrated only weak binding to the cells. The monoclonal IgGclones 0072 and 0073 showed potent binding to cytokine activated CD4+ Tcells with maximal binding observed at concentrations of 1 ug/ml orlower. The corresponding Fab clones (0087 and 0088) bound with similarpotency to cytokine activated cells, although slightly lower MFI valueswere recorded at binding saturation. The greater observed MFI values ofIgG treated compared with Fab treated cells was expected since thesecondary (detecting) antibody (anti-IgG heavy and light chain) bound toboth Fab and Fc parts of the full-length IgG antibody clones, but onlyto Fab parts of Fab clones (Table 9).

TABLE 9 Mean fluorescence intensity (MFI) values are shown forIL-12/IL-18/TL1A activated cells on day 5. Cells are gated on singlets,live and CD4+ T cells. Antibodies or Fab fragments Concentration used inassay MFI 0072 1 μg/ml 1300 0073 1 μg/ml 1300 0077 1 μg/ml 100 0087 1μg/ml 500 0088 1 μg/ml 500 0091 1 μg/ml 100

Functional Assay:

CD4+ T cells were isolated from Buffy Coats through magnetic beadseparation using CD4 Rosettesep (Stem cell technologies) and Histopaque(Sigma). T cells (2×10⁵ cells/well in a 96-well plate) were activatedfor 5 days with 2 ng/ml IL-12, 50 ng/ml IL-18 and 100 ng/ml TL1A(Flag-HIS-TEV-TL1A; Novo Nordisk) with and without anti-TL1A (1000ng/ml; MAB7441; RnDSystems) and DR3 mAbs or DR3Fabs (5 or 10 μg/ml) TheDR3 mAbs used were 0072, 0073 and 0077. The DR3Fabs used were 0087, 0088and 0091. Cells were pulsed with [³H]thymidine after 5 days ofactivation and harvested 16 h later. Alternatively cells were activatedfor 3 days and subsequent production of cytokines was measured (seeexamples 6, 15, 18).

TABLE 10 T cell assay: CD4+ T cells from healthy donors activated withIL-12/IL18 + TL1A Inhibition of cytokine production (TNFalpha)represented as IC50 values (shown as nM) and as max percentageinhibition: Two representative donors shown together with average of alltested donors. donor 1 donor 2 average average Fab Format of IC50 % IC50% IC50 % ID Fab nM inhibition nM inhibition n: nM inhib 00228 IgG4-Cys,hz 0.300 92.20 0.170 82.30 3 0.189 89.27 00123 IgG4-Ser, 0.301 83.500.320 75.90 22 0.208 79.64 chimeric 00124 IgG4-Ser, 1.112 52.30 1.20541.30 4 1.329 69.29 chimeric 00130 murine Fab 0.539 84.40 1.766 78.00 50.642 82.73 00143 IgG4-Ser, 0.148 76.70 0.074 66.00 5 0.125 70.03chimeric 00152 IgG4-Ser, 0.183 73.50 1 0.183 73.50 chimeric 00230IgG4-Ser, hz 27.74 46.00 7.282 68.00 3 15.34 55.66 00231 IgG4-Ser, hz31.73 56.00 14.91 72.00 3 17.97 60.15 00158 IgG4-Ser, hz no inhib 1.24no inhib 0.75 8 no inhib −1.80 00148 IgG4-Ser, no inhib 9.50 no inhib−8.50 2 no inhib 0.50 chimeric 00122 IgG4-Ser, no inhib 3.90 no inhib10.00 4 no inhib 6.95 chimeric

T cell proliferation was increased 3-fold by co-stimulation with TL1A.This TL1A-dependent increase was blocked by co-incubation with theanti-TL1A neutralizing control antibody. All anti-DR3 mAbs as well asthe Fab 0091 slightly inhibited proliferation at 10 μg/ml, while theFabs 0087 and 0088 completely blocked proliferation even with the lowestconcentration used in the assay. In conclusion, Fabs 0087 and 0088 arevery efficient in downregulating the TL1A-dependent co-stimulation ofCD4-positive T-cell proliferation.

Example 9

Cloning and Sequencing of Mouse 27F16A1, 27F44A2 and 28F26A3 mAbs.

Murine heavy chain and light chain sequences for anti-DR3 antibodieswere cloned from the hybridomas: 27F16A1, 27F44A2 and 28F26A3. TotalRNA, extracted from hybridoma cells using the RNeasy-Mini Kit fromQiagen, was used as templates for cDNA synthesis. cDNA was synthesizedin a 5′-RACE reaction using the SMARTer™ RACE cDNA amplification kitfrom Clontech. Subsequent target amplification of HC and LC sequenceswas performed by PCR using Phusion High-Fidelity PCR Master mix(Finnzymes) and the universal primer mix (UPM) included in the SMARTer™RACE kit as a forward primer.

PCR products were separated by gel electrophoresis, extracted using theGFX PCR DNA and Gel Band Purification Kit from GE Healthcare LifeSciences and cloned for sequencing using a Zero Blunt TOPO PCR CloningKit and chemically competent TOP10 E. coli from Invitrogen. Colony PCRwas performed on selected colonies using an AmpliTaq Gold Master Mixfrom Applied Biosystems and 13uni/M13rev primers. Colony PCR clean-upwas performed using the ExoSAP-IT enzyme mix (USB). Sequencing wasperformed at Eurofins MWG Operon, Ebersberg, Germany using either T3/T7sequencing primers.

Sequences were analyzed and annotated using the Vector NTl program. Allkits and reagents were used according to the manufacturer'sinstructions. From the hybridomas, 27F16A1 and 27F44A2, a single uniquemurine LC type kappa and a single unique murine HC, subclass IgG1, wasidentified (SEQ ID NOs 8-11). From 28F26A3 a single unique murine LCtype kappa and a single unique murine HC, subclass IgG2a was identified(SEQ ID NOs 12-13). Leader peptide sequences are not included.

BLAST Searches

The translated anti-DR327F16A1, 27F44A2 and 28F26A3 VL and VH amino acidsequences were used as query sequences. BLAST searches were performedagainst sequences in the GeneSeqP patent database (an internal databasewith imported sequences from external databases but with no access forexternal parties) using the BLASTp program. Out of the 100 highestidentity scores the highest identity score for VH's was 87.4 (28F26A3)and for VL's it was 97.3 (27F44A2). In conclusion, the VH and VLsequences for anti-DR3 represent novel sequences.

Example 10 Evaluation of Antibody Binding by Flow Cytometry

Human CD4-positive T cells were purified from Buffy coats using CD4Rosettesep (Stem cell technologies) and Histopaque (Sigma). T cells werestimulated with CD3/CD28 Dynabeads (Invitrogen) at a 1:1 ratio for 4days in T75 flask at 2 mio CD4 T cells/ml in RPMI supplemented with 10%fcs. Beads were removed using a magnet, and cells were washed twicebefore titrating in antibodies on 500.000 cells/staining. Bindingsaturation was measured after incubation with goat-anti-mousephycoerythrin (Jackson ImmunoResearch) and cells were subsequentlyanalysed by flow cytometry on a BD LSRII. All antibodies reachedsaturation at 3 μg/ml. Median Fluorescence Intensity are given in table11, while staining index (MFI Ab/MFI Isotype) is shown in table 12. Abs0070, 0072, 0073 and 0076 demonstrated stronger binding than 0071 and0077.

TABLE 11 Median Fluorescence Intensity Ab Median Fluorescent Intensity0070 2329 0071 406 0072 2650 0073 2844 0076 2645 0077 645 IgG1 isotype222

TABLE 12 Staining Index Ab Staining index 0070 10.49 0071 1.83 007211.94 0073 12.81 0076 11.91 0077 2.91 IgG1 isotype 1

An aliquot of all antibodies were conjugated using the Phycoerythrinlabelling kit lightening link (Innova Biosciences). CD4 T cells werestimulated as described above and beads were removed using a magnet,before washing the cells. Unlabelled antibodies were titration from 0-50μg/ml final concentration and added to the cells before incubated at 4°C. for 30 min. Cells were washed and directly labelled antibody is addedat 2 μg/ml. After 30 min incubation, cells were washed and run on theflow cytometer. All combinations of labelled and unlabelled antibodywere tested, and the Median Fluorescence Intensity recorded. Unlabelledantibodies competing for binding of the labelled antibodies are groupedas binding the same bin.

Only abs 0070, 0072, 0073, 0076 were able to stain the activated T cellssufficiently to bin them. As depicted in Table 13 0070, 0072, 0073, 0076were able to compete for each other, and thus are grouped in the samebin. Most likely these mAbs bind the same epitope, overlapping epitopesor cannot bind simultaneously due to steric hindrance.

TABLE 13 “Bin 1” antibodies Bin 1 0070 0072 0073 0076

Example 11 SPR Analyses of the Antibodies and Fab Fragments Binding toDifferent Domains of DR3

The extracellular moiety of DR3 is located at the aminoterminal portionof DR3 and is comprised of four TNF receptor cysteine-rich domains.Several fusion proteins were expressed and purified which containedparts of the extracellular domain of DR3 of increasing length fused tothe Fc domain of a human IgG4. The DR3 proteins used for SPR analysishad been purified to exclude oligomerized material. These proteins wereimmobilized to a CM5 sensor chip using amine coupling chemistry. Bindingof anti-DR3 antibodies termed 0070, 0072, 0073 and 0076, to thedifferent DR3-Fc fusion proteins was tested by means of plasmon surfacebinding measurements on a Biacore T100 instrument (GE Healthcare). Allof these antibodies inhibit binding of TL1A to DR3 in a FACS assay.These 4 antibodies bound to recombinant DR3 (CRD1)-Fc protein thatcontained only the aminoterminal CRD (SEQ ID NO 2) comprised of aminoacids 25-71 of DR3. Another antibody 0077, that did not inhibit TL1Abinding to DR3, did not bind to DR3 (CRD1)-Fc (SEQ ID NO 2) but bound toDR3 (CRD1+A1)-Fc (SEQ ID NO 3), which contains amino acids 25-90 of DR3,indicating that its epitope or at least part of its epitope is locatedbetween amino acids 77 to 90.

The same analysis was also performed for some of the Fab fragments. Fabfragments 0123, 0124, 0130, 0143, 0152, 0219 and 0228 bound all toDR3(CRD1)-Fc (SEQ ID NO 2)

Binding of Fab fragments 230 and 231 to DR3-Fc was also tested by SPR.Both Fab bound to DR3 (ECD)-Fc (SEQ ID NO 7) that contained the completeextracellular domain of DR3, but not to DR3 (CRD1)-Fc (SEQ ID NO 2) norDR3 (CRD1+A1)-Fc (SEQ ID NO 3), indicating that these two prior art Fabfragments do not bind to the DR3 CRD1 domain.

Measurement of Binding Kinetics for Anti-DR3Fab Fragments by SPR

The experiments were performed on a Biacore T200 instrument (GEHealhtcare) and analysed with the Biacore T200 Evaluation Software. Antihuman IgG antibodies (human IgG capture kit from GE Healthcare) wereimmobilized on all flow cells of a CM5 sensor chip according tomanufacturer's instructions. HBS-EP (GE Healthcare) with 0.1% humanserum albumin was used as running buffer. DR3(CRD1)-Fc (SEQ ID NO 2) wascaptured on the chip at low surface densities between 14 and 45 RU. Fabfragments were diluted to concentrations between 90 and 0.037 nM andsubsequently injected for 360 s. The standard dissociation time was 900s, however due to the high affinities, extended dissociation times of upto 13 000 s were applied for the highest concentrations to determinecorrect k_(d) values. Binding curves were measured at 25° C. with a flowrate between 30 and 60 μL/min.

Regeneration was performed with 3 M CaCl₂ for 2×20 s. The raw data weredouble referenced by subtraction of the signals from a reference flowcell without captured ligand and a buffer blank. Determination of thekinetic parameters was performed by fitting with a 1:1 binding modelusing the Biacore T200 Evaluation Software (GE Healthcare). Averagevalues obtained from 3 different surface densities of DR3(CRD1)-Fc (SEQID NO 2) were calculated and are stated in table 14

TABLE 14 K_(D) values Fab k_(a) [M⁻¹s⁻¹] k_(d) [s⁻¹] K_(D) [M] 1247.52E+05 2.01E−04 2.67E−10 130 1.74E+06 2.08E−04 1.20E−10 143 3.49E+065.10E−06 1.47E−12 219 3.20E+06 3.06E−05 9.55E−12 228 3.97E+06 3.19E−058.05E−12

Fab Competition Assay

The experiments were performed on a Biacore T200 instrument (GEHealthcare). Anti human IgG antibodies (human IgG capture kit from GEHealthcare) were immobilized on all flow cells of a CM5 sensor chipaccording to manufacturer's instructions. HBS-EP (GE Healthcare) with0.1% human serum albumin was used as running buffer. DR3 (ECD)-Fc (SEQID NO 7) was captured at surface densities between 700 and 1200 RU. Fabfragments were used at concentrations of 20 μg/mL. A dual injection of 2Fab fragments was performed for 300 and 150 s, respectively, followed byregeneration with 3 M CaCl₂ for 2×20 s. Binding responses of the Fabfragments were analysed with Scrubber (BioLogic Software).

Fab fragments, which could not bind simultaneously to DR3 (ECD)-Fc, wereconsidered to belong to the same epitope bin. As shown in table 15, Fabfragments 124, 130, 143 and 228, which are all directed against the CRD1domain, fall into the same epitope bin. Likewise Fab fragments 230 and231, which have the same CDR domain, also belong to the same epitopebin, different from the CRD1. Fab fragments 148 and 163 do not competewith any of the other Fab and are therefore falling into separateepitope bins. Fab fragment 148 does not compete with 124, 130, 143 and228, when injected as 2. antibody, indicating that it belongs to adifferent bin. However, when 148 is bound first, neither 130, 143 or 228do bind any longer, suggesting that there either is sterical hindranceor conformational change of DR3-Fc, that precludes binding of the otherFab fragments.

TABLE 15 Fab competition for binding DR3-Fc measured by SPR. 1. Fab\2.Fab 124 130 143 148 163 228 230 231 124 − − − + + − + n.t. 130 − − − + +− + n.t. 143 − − − + + − + n.t. 148 n.t. − − − + − + + 163 n.t. n.t. + +− + + + 228 n.t. n.t. − + + − + + 230 + + + + + + − − 231 n.t.n.t. + + + + − − +: simultaneous binding of both Fab ; −: no binding of2. Fab; n..t.: not tested

Competition Assay

As shown in table 16, Fab fragments 228, 231, 230 and 148 were testedfor ability for block Mab binding of 0107 (Mab of 0228) and 0121(Mab of148) binding to DR3 expressing cells on FACS. Fab fragments 230 and 231can not compete any of Mabs and can bind simultaneously with Mab 017 andMab 0121.

TABLE 16 Competition assay performed by FACS Competition assay Fabstested for blocking of binding Mab using up to 1:10000 excess Fab. MABID 0107 0121 Fab ID (murine Mab 0228) (murine Mab of 0148) 0228 Inhibitsfrom No inhibition molar ratio Mab/Fab 1:1 0230 No inhibition Noinhibition 0231 No inhibition No inhibition 0148 Inhibits from Inhibitsfully from molar ratio molar ratio Mab/Fab 1:100 Mab/Fab 1:3

Example 12 Mapping the Epitope of Fab-0228 by Mutagenesis of DR3

In order to determine which amino acids in the CRD1 of human DR3 areresponsible for interaction with the -0123 Fab, a few DR3 mutants weregenerated. The three mutations, R29Q, I43N and L45V were made bysite-directed mutagenesis (Quikchange, Stratagene). The mutated DR3expression plasmids and the NFkappaB-Luc reporter plasmid weretransiently transfected into HEK293 cells and the cells were shortlypreincubated with anti-DR3 anti-Fab -0228 and subsequently stimulatedwith human TL1A for four hours. The luciferase activity was assayed byusing Steady-GLO (Promega) and monitored on a TopCount NXT (PerkinElmer). The R29Q was neutralized slightly better than the humanwildtype, whereas both I43N, L45V and the combined mutants I43N/L45Vwere neutralized much less efficiently, see table 17.

The anti-Fab -0123 was also tested and the ranking was similar to the-0228; R29Q>WT>L45V>I43N>I43N-L45V. Thus, residues I43 and L45 are veryimportant for maintaining the binding affinity of 0228 to DR3. Theseresidues are therefore most likely part of the binding epitope. Incontrast, R29 does not seem to be essential for the 0228 binding to DR3since the affinity is maintained and even slightly improved when thisresidue is mutated. However, since this position affects the 0228binding it is of structural importance for the binding epitope.

TABLE 17 Fabs tested on the transiently transfected primate DR3 reportercells, IC50 of Fab upon stimulation by TL1A Fab ID Human DR3 R29Q I43N

 45V I43N/L45V 0227-0000-0228 645 pM 372 pM 68 nM

 nM 149 nM

indicates data missing or illegible when filed

Example 13 Anti-DR3 Humanization

The sequence of 0072 was obtained from cloning of the hybridoma 27F44A2.All numbering used in this example refers to the Kabat numbering scheme.

>0072VH (CDRs marked with bold) (SEQ ID NO 10)EVKLVESGGGLVKPGGSLKLSCSASGFAFSNYDMSWVRQTPEKRLEWVAAFSSDGYTFYPDSLKGRFTISRDNARNTLYLQMSSLGSEDTALYCCARHADYANYPVMDYWGQGTSVTVSS >0072VL (CDRs marked with bold)(SEQ ID NO 11)DIVLAQSPASLLVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTMLELKR

A sequence analysis of 0072 reveals a free cysteine at position 91 ofthe heavy chain, that must be removed during the humanization.

A 3D model of 0072 was build using standard techniques in MOE [availablefrom www.chemcomp.com] and all residues within 4.5 Å of the effectiveCDR regions (VH: 31-35B, 50-58, 95-102; VL: 24-34, 50-56, 89-97) aredefined as mask residues. Mask residues are all potentially importantfor sustaining the binding in the CDRs.

The mask residues includes positions 2, 4, 27-37, 47, 50-59, 69-71, 78,91-103 for the heavy chain and positions 3-5,7,23-36, 46-60, 62, 65, 67,69-71, 87-98 for the light chain.

Using germline searches and manual inspection VH3_(—)13 and JH4 wereidentified as an appropriate human germline combination for the heavychain and VKI_(—)02 and JK4 were identified as the appropriate humangermline combination for the light chain.

The humanization can now be performed with the following rules:

-   -   Residues outside the mask are taken as human.    -   Residues inside the mask and inside the Kabat CDR are taken as        murine.    -   Residues inside the mask and outside the Kabat CDR with        mouse/germline consensus are taken as the consensus sequence.    -   Residues inside the mask and outside the Kabat CDR with        mouse/germline difference are subject to potential back        mutations.

Grafting the effective CDR regions of 0072 into the germlines forms thebasic humanization construct of 0072, hz0072.

1. >hz0072VH (SEQ ID NO 28) 2.EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQATGKGL EWVSAFSSDGYTFYP 3.GSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARHADYANYP VMDYWGQGTLVTVSS 4.5. >hz0072VL (SEQ ID NO 29) 6.DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPG KAPKLLIYLASNLES 7.GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPLTFGGG TKVEIK

The only differences compared to the murine CDRs are in CDR_H2

Any discrepancy between 0072 and hz0072 in a mask residue will create apotential backmutation and the list includes

hz0072VH: T28A, S49A, Y91C

hz0072VL: Q3V, M4L, TSM, S60A

In order to investigate all potentially humanized mAbs all combinationsof the above mutants have to be produced.

The Fab proteins listed in Table 18 were expressed in HEK293 6E cellsand expression levels of each variant were compared as evaluated bySDS-PAGE gel. Results in Table 18 showed that all the variants thatcontained the germline Tyr at site-91 of heavy chain (compound 0169,1070 and 0171) had dramatically improved expression level comparing tothose that contained other replacement, either Cys (compound 0172) orSer (compound 0173 or 0174), at this site. Also from Table 18, humanversion of Met or murine version of Leu at site-4 of light chain did nothave obvious effect on the expression level. This indicated that Y91 inthe heavy chain is critical to the improvement of expression level ofthe fully-grafted version of 0072.

TABLE 18 Comparison of expression level of Fab by variants of anti-DR3Fab that had mutations on site-91 of heavy chain or site-4 of lightchain. Compound Mutation site Expression name HC_91 LC_4 level 0169 Y91M4L ++++ 0170 Y91 M4 ++++ 0171 Y91 M4 ++++ 0172 Y91C M4L + 0173 Y91SM4L + 0174 Y91S M4 + The variants were generated on the humanizedanti-DR3 0072 with backmutations of HC_T28A_S49A and LC_Q3A_T5A_S60A.HC_Y91 is the humanized version; Y91C is the murine version; Y91S is tomutate Y91 to an irrelevant amino acid Ser. LC_M4 is the humanizedversion; LC_M4L is the murine version. Expression level was evaluated bySDS-PAGE gel; “++++” represents high expression level of >50 mg/L, and“+” represents poor expression level.

Selection of Back Mutation

The potential advantage of inserting a back mutation were evaluatedbased on expression, potency and affinity screening as well as bybiophysical characterization.

Expression pattern: The expression levels of various humanized versionsof 0072 including various back mutations (BM's) were evaluatedtransiently in HEK293-6E (see example 3 for transient expression inHEK293-6E).

In addition to the hz0072 the following BM's were evaluated individuallyregarding expression:

LC: T5A and 560A

HC: T28A and S49A

The experiment was performed in triplicates. Cells were grown 5 dayspost transfection. Media were harvest and the expression levels weremeasured by forteBlas Octet using protein G sensors.

The result show that the BM S49A on HC resulted in a ˜2 fold increasecompared to hz0072 expression. Neither of the other BM had thecapability to increase expression level (Table 19).

TABLE 19 Relative expression level of the heavy chain (HC) with backmutation and the light chain (LC) with back mutation and compared to thewild type (WT) that is set as 100% expression level. Average (%) Stdevhz0072 100 0.06 hz0072 91.7 6.02 T28A(VH) hz0072 251.2 4.58 S49A(VH)hz0072 T5A(VL) 45.7 1.26 hz0072 63.6 2.17 S60A(VL)

Biophysical Characterization

The thermostability of the humanized Fabs was measured usingdifferential scanning fluorimetry (DSF). DSF was performed using a MyiQReal-Time PCR detection system (Biorad Laboratories, Inc). Samples inPBS at pH 7.4 at a final concentration of 0.3 mg/ml were placed insealed 96 well PCR plates and 2000 times diluted stock solution of thedye Sypro Orange, which was used to monitor the protein unfoldingtransition. The fluorescence intensity was measured withexcitation/emission wavelength: 480/575 nm. Table 20 shows thethermostability of the humanized Fabs. The results show the two Fabswith back mutations (228 and 229) had the same thermostability (Tm) at71° C., whereas the humanized Fab without back mutations (227) had adecreased thermostability at 66° C.

TABLE 20 T_(m) (denaturation temperature) values are shown for thehumanized Fabs. Fab _(m) (° C.) hz0072 (227) 6 hz0072 S49A(VH) (228) 1hz0072 S49A(VH) 1 S60A(VL) (229)

Selection of IgG4 Backbone Using Biophysical Evaluation

The IgG1 and IgG4 isotypes of the humanized Fabs were measured at 3mg/ml in PBS at pH 7.4 using Bioanalyser to look at the fragmentationpatterns. An Agilent 2100 Bioanalyzer was used to look at fragmentationpatterns. The kit was Agilent Protein 230, and the marker used wasProtein 230 Ladder with 7 peaks. Non-reduced samples were prepared with2 μl sample, 2 μl Mill-Q water, 2 μl sample buffer and 1 μl 0.5 MN-ethylmaleimide (NEM dissolved in CH3CN). All samples were heated to100° C. for max. 5 minutes.

Table 21 shows the Bioanalyser data of the integrated amount of free LCfor the Fabs at non-reduced conditions. The results show that all theIgG4 Fabs all have less free LC in non-reduced conditions compared tothe IgG1 Fabs.

TABLE 21 Integrated peak values of free LC in non-reduced conditions forIgG1 and IgG4 humanized Fabs. Fab gG1 gG4 hz0072 1.9 .0 hz0072S49A(VH).6 .6 hz0072S49A(VH) .2 .7 S60A (VL)

After extensive potency and affinity screening, expression analysis andstability studies as described above the final humanized candidate wasselected as the IgG4 version of hz0072VH S49A, hz0072VL with thefollowing sequence:

8. >hz0072VH_S49A (SEQ ID NO 16) 9.EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQATGKG LEWVA AFSSDGYTFYP 10.GSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARHADYANY PVMDYWGQGTLVTVSS11. >hz0072VL (SEQ ID NO 17) 12.DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKP GKAPKLLIYLASNLES 13.GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPLTFGG GTKVEIK

This candidate has no free cysteine in the variable domain.

Example 14 Purification of Recombinant Fab Fragments:

Recombinant Fab fragments were purified by chromatography steps using anAkta Explorer FPLC system (GE Healthcare). Cell culture supernatantscontaining recombinant mouse Fab were diluted in order to reduce theconductivity below 2 mS. Depending on the theoretical pH of the Fabfragment, either anion or cation exchange chromatography was performedusing MonoQ or MonoS columns (GE Healthcare). Fab fragments were boundto the column in a buffer of low ion strength at pH values between 5 and8.5 and eluted with a salt gradient up to 1 M NaCl. Gel filtration witha Superdex200 column (GE Healthcare) and PBS as buffer was performed toremove oligomeric Fab complexes.

Chimeric Fab fragments, containing the constant part of human kappaimmunoglobulin light chain, were purified by affinity purification withKappaSelect (GE Healthcare) according to manufacturer's instructions,followed by gel filtration to remove high molecular weight complexes.

Generation of Fab by Papain Cleavage of mAb

Fab fragments were generated by cleavage of mAb using the Pierce FabPreparation Kit (Thermo Scientific) according to manufacturer'sinstructions. In some cases, gel filtration with a Superdex200 column(GE Healthcare) and PBS as buffer was performed to remove high molecularweight forms.

Example 15

Cytokine Release from or Proliferation of Primary Human T Cells.

CD4+ T cells isolated from Buffy Coats through magnetic bead separationusing CD4 Rosettesep (Stem cell technologies) and Histopaque (Sigma)were stimulated in the absence of TCR activation, with cytokines IL-12(2 ng/ml), IL-18 (50 ng/ml) with and without 100 ng/ml TL1A(Flag-His-TEV-TL1A; Novo Nordisk). T cell proliferation and cytokinerelease was measured. Supernatants from CD4+ T cells (2×10⁵ cells/well)stimulated for 48 h were harvested and analysed for cytokine release byBioplex. T cell proliferation was measured after 5 days.

IL-12 (2 ng/ml), IL-18 (50 ng/ml) and 100 ng/ml TL1A (Flag-His-TEV-TL1A;Novo Nordisk). treated CD4+ T cells demonstrated 6-fold increased T cellproliferation compared to IL-12/IL-18 treated T cells. The 0123 and 0124Fabs totally blocked the induced T cell proliferation already at aconcentration of 0.16 μg/ml of the Fab while the corresponding mAb 0072and 0108 did not affect T cell proliferation (FIG. 8).

The released cytokines IL-6, IFNγ, GM-CSF and TNFα were all inducedafter 48 hours by TL1A by IL-12/IL-18 treated CD4+ T cells. TNFα wassignificantly (p=*) blocked by the anti-DR3Fab 0124, compared to a DR3binding non-blocking Fab 0091 (Table 22).

TABLE 22 +TL1A (fold upregulation +TL1A + anti DR3 Fab (fold compared toIL12/IL18 downregulation compared to Cytokines activated T cells)IL12/IL18 activated T cells) IL-6  16 (p = 0.051) 13.3 (p = 0.08) IFNγ1.2 (p = 0.025)  1.1 (p = 0.08) GM-CSF 7.5 (p = 0.04)  7.9 (p = 0.08)TNFα 4.4 (p = 0.0007 =   5 (p = 0.025)

Example 16 Conjugating a Lipid Moiety to a Fab

NAP-25 columns, cat#17-0852-02 from GE HealthcareHitrap Q-sepharose FF column (code: 17-5156-01) from GE HealthcareTSPP: Triphenyl phosphine-3,3′,3″-trisulfonic acid trisodium salthydrate, cat#39538, from Alfa AesarTDSPP: Bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt,cat#151888-20-9, from Strem ChemicalsTEA: triethanolamine, product nr.: 90279 from SigmaEthyleneglycol, cat#1.00949.1000 from MERCKNaCl: Code: 207790010 from Acros OrganicsEDTA, disodium salt, dihydrate cat#SC-29092 from ChemCruzPBS Tablets, cat#18912-014 from GIBCO

Vivaspin 20, 10000 MWCO, PES membrane, cat#VS2001 from Sartorius PD10G-25 columns, cat#17-0851-01 from Ge Healthcare

Abbreviations

CV: column volumesFLD: fluorescence detectionMQ: MilliQ water (highly purified water)m/z: mass to charge ratioMS: mass spectrometryM+H: mass of a singly protonated speciesHPLC: high pressure liquid chromatographyRP: reversed phaseLC-MS: liquid chromatography—mass spectrometryNMR: nuclear magnetic resonance spectroscopyrt or RT: room temperatureBoc: tert butyloxycarbonylO-t-Bu: tert butyl estert-Bu: tert butylDCM: dichloromethane, CH₂Cl₂, methylenechlorideDIC: diisopropylcarbdiimide

DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide

DMSO: dimethylsulfoxideFmoc: 9H-fluoren-9-ylmethoxycarbonylLys(Mtt)-OH: (S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-amino-hexanoicacidThx: trans-4-aminomethylcyclohexancarboxylic acid

NMP: N-methylpyrrolidin-2-one

OEG: (2[2-(amino)ethoxy]ethoxy)acetic acidTFA: trifluoroacetic acidTHF: tetrahydrofuranTIS: triisopropylsilaneTNBS: trinitrobenzene sulfonic acidHC: heavy chainLC: light chainwt: wild typeAmino acid abbreviations follow IUPAC conventions.Buffer abbreviations follow Stoll, V. S, and Blanchard, J. S., Methodsof Enzymology, 182, 1990, Academic Press, 24-38.

Conjugations, General Considerations

Fab's for conjugation with hydrophobic protraction groups wereconstructed with an unpaired cysteine at or near the C-terminus of theheavy chain (HC). This cysteine was intended for conjugations withthiophilic alkylation reagents bearing eg. maleimide or halo-acetylgroups. The Fab's were expressed as molecules with a disufide bondedcysteinylation to the mutated Cys. In order to free the mutated Cys forreaction with alkylation reagents, it was reduced using a phosphinereagent. After reduction, the protein was separated from the reducingreagent and subjected to alkylation by adding the alkylation reagent.Finally, the conjugate was purified by standard low pressurechromatography.

4-(1H-Tetrazol-16-yl-hexadecanoylsulfamoyl)butanoyl-OEG-yGlu-γGlu-γOEG-N^(ε)(C(O)CH₂Br)Lys-OH(albumin binder I):

Compound I was synthesised on solid support according scheme 1, in 1 mMscale using standard Fmoc-peptide chemistry on an ABI433 synthetizer(Applied Biosystems). The peptide was assembled on a Fmoc-Lys(MTT)-Wangresin using Fmoc-OEG-OH and Fmoc-Glu-OtBu protected amino acids.4-(16-1H-Tetrazol-5-yl-hexadecanoylsulfamoyl)butyric acid was manualcoupled using DIC/NHS in DCM/NMP, 2 eq. over night, TNBS test showed thereaction to be completed. The resin was then treated with 50 mLDCM/TFA/TIS/water (94:2:2:2) in a flowthrough arrangement until theyellow colour disappeared, ˜20 min. followed by washing and neutralizingwith DIPEA/DMF. Bromo acetic acid (4 mM) in DCM/NMP (1:1) was activatedwith a 1 mM mixture of NHS and DIC, filtered and added to the resin withaddition of further 1 mM of DIPEA. After 1 hr the reaction wascompleted. The resin was treated with 80 mL TFA/TIS/water (95:2.5:2.5)for 1 hr. Evaporated with a stream of N₂, precipitated by addition ofEt₂O and washed with Et₂O and dried. Crude product was purified onpreparative HPLC (2 runs), with a gradient from 30-80% 0.1 TFA/MeCNagainst 0.1% TFA in water. Fractions were collected and lyophilized with˜50% MeCN affording compound I.

TOF-MS: mass 1272.52 (M+1)

The above example shows that it is possible to prepare albumin bindercompound I in high purity using solid phase synthesis.

Conjugation of Fab 0120 to Albumin Binder I

Fab 0120 (25 mg, 0.53 umol, 4.2 mg/ml) in PBS buffer was mixed with asolution of triphenylphosphine-3,3′,3″ trisulfonic acid trisodium salthydrate (TSPP, 40 mg/ml, 350 ul, final conc. 3.7 mM) in 20 mMtriethanolamine, 2 mM EDTA, pH 8.5. The reaction mixture was incubatedat r.t. for a period of 150 min. Small molecular weight substances werethen removed by buffer exchange on three NAP-25 columns equilibrated andeluted with 20 mM triethanolamine, 2 mM EDTA, 400 mM NaCl, pH 8.5. Thebuffer exchanged reduced Fab-solution was then mixed with a solution ofalbumin binder I (490 ul, 16 mM, final conc: 0.8 mM) in 20 mMtriethanolamine 2 mM EDTA, 400 mM NaCl, pH 8.5. The reaction mixture(approx. 9.5 ml) was incubated at r.t. for a period of 20 hours and thendiluted with 10 ml of buffer 20 mM triethanolamine, 10% ethylenglycol,pH 8.0. Subsequently, the mixture was buffer exchanged on four NAP25columns equilibrated with 20 mM triethanolamine, 10% ethylenglycol, pH8.0. The buffer exchanged material was loaded to a 5 ml HitrapQ-sepharose FF column equilibrated with 20 mM triethanolamine, 10%ethylenglycol, pH 8.0 using an Akta Puifier 100 system (GE Healthcare).Unbound material (incl. the inconjugated Fab) was washed out using thisbuffer and the product was eluted using a linear gradient of 20 mMtriethanolamine, 10% ethylenglycol, 1 M NaCl, pH 8.0 over a period of1.5 hours. The product eluted at approx. 380 mM NaCl with a symmetricpeak profile. Relevant fractions were pooled, concentrated byultra-filtration a using Vivaspin 20 devices at 3500 g (10000 MWCO,Sartorius) and finally buffer exchanged to PBS buffer. The finalisolated product was 11.9 mg (48%), at a concentration of 1.7 mg/mldetermined by A280 measurement. SDS-PAGE and MS analysis confirmed theidentity of the product (found m/z 49919 (M+H), calc. 49917 (M+H)).

The above example shows a successful, site-specific conjugation of theFab with the albumin binder I. The conjugation gives rise to a productin good yield and high purity.

Conjugation of Fab's to Albumin Binder II-VI

Albumin binders II-VI has the structures as shown below

Conjugation of albumin binders I-VI followed the same procedure asdescribed for compound I above. In the case of albumin binders IV andVI, the compounds were not fully soluble before addition to theFab-solution. In some cases organic solvent was added to facilitatesolubilisation. Due to the lower number of acidic groups on albuminbinders II-VI, the conjugates did not bind to an AIEX column unless pHwas increased to 8.6. In the case of albumin binders II-VI, the puremono-substituted product was contaminated with varying amounts ofmultiply modified Fab. These were difficult to remove by ion exchangechromatography.

The identity of all conjugates were confirmed by SDS-page and LC-MSexample shown in table 23. The purity was determined to be >90% forconjugates with II, III and V, while conjugates with IV and VI containedapprox. 30% of an un-conjugated Fab-based impurity.

TABLE 23 LC-MS data of two different Fab's conjugated to various albuminbinders, compared to the calculated mass. Conjugate with Compound m/zfound m/z calculated albumin binder number Fab number (M + H) (M + H) II0190 0120 49629 49629 III 0192 0120 49657 49657 IV 0193 0120 49427 49528V 0191 0120 49543 49543 VI 0194 0120 49550 49550 II 0243 0228 4876448765 III 0240 0228 48792 48793 IV 0242 0228 48663 48664 V 0244 022848678 48679 I 0241 0228 49053 49053

The above example shows that a variety of different albumin binders canbe conjugated to Fab's of both IgG1 and IgG4 origin, in good yield andpurity.

The above example shows that a variety of different albumin binders canbe conjugated to Fab's of both IgG1 and IgG4 origin, in good yield andpurity.

Conjugation of Various Fab's with Albumin Binder I

The following Fab's were conjugated: 0118, 0119, 0127, and 0147 (Seetable 25). These Fab's contained an unpaired cysteine residue at theC-terminal, or in the case of 0157, close to the C-terminal. The Fab'swere derived from IgG1 or IgG4 and correspond to different clones allbinding the DR3 receptor. When reacted according to the method describedabove, conjugates were formed in good yield according to LC-MS data. Thefollowing MS data were obtained (Table 24)

TABLE 24 LC-MS data of various Fab's conjugated with albumin binder I,compared to the calculated mass Conjugate with Fab m/z found (M + H) m/zcalculated (M + H) 118 49947 49947 119 49408 49408 127 49712 49712 13449975 49975 147 49409 49409 157 50169 50169

The above example shows that a variety of Fab's can be conjugated todifferent albumin binder molecules in good yield and purity.

TABLE 25 List of anti-DR3 Fab albumin binder conjugates for PK studies.Con- Conjugated Internal Fatty acid jugated compound Albumin binderstructure Number Length to Fab number

I Not applicable 0120 0228 0118 0119 0127 0147 0153 0241   0189     0166

II C18 0120 0228 0190 0243

V C18 0120 0228 0191 0244

III C20 0120 0228 0192 0240

IV C20 0120 0228 0193 0242

VI Not applicable 0120 0194 A note on nomenclature: This table usesfour-number identification (e.g. 0228). However, different nomenclaturecan be observed for the same compound. For example compound 0228 is alsobe named 0227-0000-0228, 0227-0228, 00228, or 228. All thesenomenclature denotes the same compound. This rule applies to allcompounds.

Example 17

The main objectives have been to characterize the pharmacokineticparameters after both iv and sc administration to DBA/1 mice, main focushave been on the terminal half-life in order to evaluate the extent ofprolongation after modifying the Fab fragment.

Study Design and Methods

Results from two studies are included in the present summary (DKPF111105and DKPF110703). Both studies were performed in male DBA/1 mice, themice were dosed either intravenously (i.v.) in the tail vein orsubcutaneously (s.c.) in the groin. Dosings were 5 mg/kg for Fab and 1mg/kg for modified Fab. A sparse sampling design were used, three bloodsamples were collected at each sampling point, samples were collectedfrom pre-dose up till 7 days after dosing, depending on the compound(e.g. when testing a Fab samples were only collected up to 7 hours afterdosing, whereas samples were collected up to 7 days after dosing whentesting a conjugated Fab).

The collected plasma samples were all analysed using an ELISA assay,briefly the plates were coated with hDR3-Fc fusion protein (SEQ ID NO7), diluted blood samples were added and the analyte (Fab or conjugatedFab) would bind to the hDR3, the bound analyte was then detected by aperoxidase labelled anti-Fab antibody, and the absorbance were measureusing standard methods. Base on the measured plasma concentrationvalues, the pharmacokinetic parameters were assessed using standardnon-compartmental methods, using the commercial available softwarePhoenix WnNonlin (Pharsight Corp.).

Results

Several different linkers were evaluated to prolong the terminalhalf-life of the Fab, based on the albumin binding technology it waspossible to increase the terminal half-life of the Fab to more than 20hours for several of the molecules (Table 26). The half-life of the Fabwas estimated to be below 1 hour, which illustrates the difference ofthe Fab compared with a conjugated Fab (Table 26) that all display amuch longer half-life.

TABLE 26 Pharmacokinetic parameters after iv of sc administration tomice. Dose T½ MRT Cl F Study Route COMPOUND NNC (mg/kg) (h) (h)(mL/h/kg) (%) DKPF111105 iv IgG1 (G1m17) chimeric Fab wt Cys0227-0000-0120-1A 5 0.8 0.2 158 — DKPF111105 iv 0120 with albumin binderlinker 0195- 0227-0000-0190-1A 1 14 17 1.7 — 0000-3007 C18+ GluDKPF111105 iv 0120 with albumin binder linker 0186- 0227-0000-0192-1A 121 25 1.9 — 0000-3007 C20+ Glu DKPF111105 iv 0120 with albumin binderlinker 0195- 0227-0000-0193-1A 1 24 24 3.0 — 0000-3007 C20+ GluDKPF111105 iv 0120 with albumin binder linker 0194- 0227-0000-0191-1A 116 18 1.8 — 0000-3032 (FGF-21 linker) C18+ Glu DKPF111105 iv 0120 withalbumin binder 0227-0000-0194-1A 1 7.2 9 4.3 — DKPF111105 sc IgG1(G1m17) chimeric Fab wt Cys 0227-0000-0120-1A 5 1.1 2.5 — 13 DKPF111105sc 0120 with albumin binder linker 0186- 0227-0000-0192-1A 1 27 37 — 310000-3007 C20+ Glu DKPF111105 sc 0120 with albumin binder linker 0195-0227-0000-0193-1A 1 23 32 — 34 0000-3007 C20+ Glu DKPF111105 sc 0120with albumin binder linker 0194- 0227-0000-0191-1A 1 19 27 — 520000-3032 (FGF-21 linker) C18+ Glu DKPF111105 sc 0120 with albuminbinder 0227-0000-0194-1A 1 32 — — 34 DKPF110703 iv 0120 with albuminbinder 0227-0000-0153-1A 1 25 30 1.1 — DKPF110703 iv 0120 wt with Cys toSer 0227-0000-0124-2A 1 0.2 0.16 183 — DKPF110703 iv IgG1 (G1m17)chimeric Fab wt Cys 0227-0000-0120-1A 1 0.4 0.31 181 — DKPF110703 sc0120 with albumin binder 0227-0000-0153-1A 1 24 40 — 55 DKPF110703 sc0120 wt with Cys to Ser 0227-0000-0124-2A 1 . — — 16 DKPF110703 sc IgG1(G1m17) chimeric Fab wt Cys 0227-0000-0120-1A 1 . — — 22 T½: terminalhalf-life, MRT: Mean Residence Time, Cl: Clearance; F: Bloavailability

Example 18 Reading Interferon-Gamma Release as a Way to DetermineAnti-DR3 Activity

Fresh human peripheral blood mononuclear cells (PBMC) were separatedfrom the blood buffy coat (307 Hospital Blood Center of PLA; Beijing,PRC) by centrifugation in Ficoll (GE Healthcare, Cat#17-5442-02) for 20min at 2000 rpm. After wash in DPBS, the cell pellet was aspirated andresuspended in pre-warmed assay media (RPMI 1640, 10% heat inactivatedFBS, 1% penicillin/streptomycin) at 1×10⁶/ml for overnight culture inflask. Non-adherent lymphocytes were then removed and used in the T cellcostimulation assay.

For costimulation assay, recombinant human TL1A (12 μg/mL) was added asa costimulator at 50 μl/well to a dry 96 well U-bottom assay plate(Corning costar 3799) that had been pre-coated, to all wells except theedge, with the primary stimuli, anti-CD3 mAb (BD Pharmingen cat#555336)at 0.3 μg/mL in a 37° C. incubator for 3 hours. The testing anti-DR3Fabs (0170, 0171, 0173, 0169 & 0118) were transferred in duplicate tothe assay plate at 50 μL/well, following an 1:3 serial titration in 96well deep-well plates (Nunc 278743) for 4× concentrations ranging from0.012 to 12 μg/mL at 1.5 mL/well. Then the assay plates were added with0.1 mL of the enriched lymphocytes at 2×10⁵ cell/well, bringing thefinal volume to 0.2 mL/well. After 40 hours of cell cultures in 37° C.incubator with 5% CO₂, 120 mL supernatants were harvested from each wellof the culture plates in order to measure cytokine secretion by IFN-γELISA kits (eBioscience Cat#88-7316-88). According to manufacturer'ssuggestions, the testing supernatants were 1:20 diluted in order tomeasure the full range of the cytokine been secreted in the T cellassay.

A number of humanized anti-DR3Fabs with (173, 170 & 169) or without(171) back mutations was evaluated for their ability to block theTL1A-costimulated cytokine secretion of primary T cells from a healthydonor's peripheral blood.

As shown in Table 27, anti-CD3 priming caused a moderate level (˜50ng/mL) of IFN-γ secretion, which was enhanced more than 3 fold (˜159ng/mL) by recombinant TL1A costimulation; whereas TL1A treatment aloneonly resulted in a minimal amount of the cytokine release (1.2 ng/mL).Under the TL1A costimulation condition, all testing humanizedanti-DR3Fabs inhibited IFN-γ secretion in dose-dependent and completemanner (IC₅₀˜0.01-0.03 ug/mL), with minimal effect on anti-CD3 priming.As the negative control identified from a number of experiments withmultiple donors, a non ligand-blocking chimeric anti-DR3 Fab 0118 showedno significant effect as expected on TL1A costimulation of Tlymphocytes. Similar results were obtained on second batch of 0171 and0170 Fabs in another experiment with a different blood donor.

TABLE 27 Effect of humanized anti-DR3 Fabs on IFN-γ secretion of TL1A-costimulated T lymphocytes from a healthy blood donor IFN-γ (ng/mL)[μg/mL] cFab-0118 hFab-0169 hFab-0170 hFab-0171 hFab-0173 0 159 ± 38 159 ± 38  159 ± 38  159 ± 38  159 ± 38  0.003 132 ± 0.2  121 ± 0.2  131± 31  140 ± 8.2  160 ± 3.7  0.01 134 ± 0.3  138 ± 34  101 ± 64  105 ±58  163 ± 0.5  0.03 158 ± 42  81 ± 26 108 ± 24  69 ± 10  95 ± 2.8 0.1151 ± 0.1  58 ± 25 46 ± 27 52 ± 12 69 ± 18 0.3 124 ± 68   31 ± 0.1  51 ±7.1  62 ± 0.3 62 ± 11 1.0 132 ± 55  55 ± 24  63 ± 4.7  40 ± 0.4  48 ±3.1 The number after ± indicates the standard deviation in thedetermination.

Example 19 Anti-DR3 Antibody Fabs Block TL 1a Costimulation of EffectorT Cells in Synovial Fluid of Rheumatoid Arthritis Patients

Humanized anti-DR3Fabs were shown to inhibit the activity of primary Tcells isolated from peripheral blood of healthy individuals in theprevious examples. Additionally, some Fab's were also evaluated fortheir ability to block the effector function of synovial T cells derivedfrom rheumatoid arthritis (RA) patients. Effector functions of T cellsinclude, but are not limited to, IFN-γ secretion which was analyzed inthis example. The levels of secreted IFN-γ were measured by the sameELISA kit as described in Example 18.

Fresh human synovial fluid (SF) cells were separated, by centrifugationfor 10 min at 2000 rpm, from the knee joint fluid of RA patients as theside product (waste) of intra-articular injection of DMARDs (BeijingUniversity People's Hospital, PRC). The cell pellet was aspirated andresuspended in pre-warmed assay media (RPMI 1640, 10% heat inactivatedFBS, 1% penicillin/streptomycin) at 1×10⁶/mL. A small sample (˜10⁵/0.1mL) of the synovial cells was examed for the presence (%) of Tlymphocyte by FACS analysis. RA/SF cell samples with >5-10% lymphocytepopulation were used in the T cell costimulation experiments.

For costimulation assay, recombinant human TL1A (12 μg/mL) was added asa costimulator at 50 μl/well to a dry 96 well U-bottom assay plate(Corning costar 3799) that had been pre-coated, to all wells except theedge, with the primary stimuli, anti-CD3 mAb (BD cat#555336) at 0.3μg/mL in a 37° C. incubator for 3 hours. The testing anti-DR3Fabs (0170,0171, 0173 & 0118) were transferred in duplicate to the assay plate at504/well, following an 1:3 serial titration in 96 well deep-well plates(Nunc 278743) for 4× concentrations ranging from 0.012 to 4 μg/mL at 1.5mL/well. Then the assay plates were added with 0.1 mL of RA/SF cells at2×10⁵ cell/well, bringing the final volume to 0.2 mL/well. After 40hours of cell cultures in 37° C. incubator with 5% CO₂, 120 mLsupernatants were harvested from each well of the culture plates inorder to measure cytokine secretion by IFN-γ ELISA kits (eBioscienceCat#88-7316-88). According to manufacturer's suggestions, theappropriate dilution factor was determined on the testing supernatantsin order to measure the full range of the cytokine been secreted in theT cell assay.

Humanized anti-DR3Fab (171) and its backbone-mutated variants (173 &170) were evaluated for their ability to block the TL1A-costimulatedcytokine secretion of synovial T cells from RA patients' knee joints.

As shown in Table 28, anti-CD3 priming induced a moderate level (˜1228pg/mL) of IFN-γ secretion, which was enhanced about 2 fold (˜2188 pg/ml)by recombinant TL1A costimulation; whereas TL1A treatment alone showedno detectable cytokine release. Under the TL1A costimulation condition,three testing humanized anti-DR3Fabs inhibited IFN-γ secretion indose-dependent and complete manner (IC₁₀₀˜0.1 ug/mL), with minimaleffect on anti-CD3 priming. As the negative control, a nonligand-blocking chimeric anti-DR3Fab 0118 showed no significant effectas expected on TL1A costimulation of T lymphocytes. Similar results wereobtained on different batches of 0171 (3 batches) and 0170 (2 batches)Fabs in five experiments with different RASF samples.

TABLE 28 Humanized anti-DR3 Fabs Inhibit TL1A-costimulated IFN-γsecretion of SF cells from a RA patient IFN-γ (pg/mL) [μg/mL] cFab-0118hFab-0170 hFab-0171 hFab-0173 0 2188 ± 255  2188 ± 255  2188 ± 255  2188± 255  0.003 2308 ± 639  1794 ± 366  1870 ± 80  1552 ± 195  0.01 2280 ±0.4   1635 ± 370  1375 ± 248  1230 ± 248  0.03 2079 ± 368  1357 ± 331 1220 ± 0.4   1127 ± 122  0.1 2103 ± 0.0   1166 ± 0.2   1143 ± 65  1131 ±82  0.3 2121 ± 75  1301 ± 0.1   1157 ± 180  992 ± 56  1 2232 ± 567  1249± 184  1232 ± 0.1   1062 ± 36 

Example 20 Epitope Mapping by HX-MS of Anti-DR3Fabs

The HX-MS technology exploits that hydrogen exchange (HX) of a proteincan readily be followed by mass spectrometry (MS). By replacing theaqueous solvent containing hydrogen with aqueous solvent containingdeuterium, incorporation of a deuterium atom at a given site in aprotein will give rise to an increase in mass of 1 Da. This massincrease can be monitored as a function of time by mass spectrometry inquenched samples of the exchange reaction. The deuterium labellinginformation can be sub-localized to regions in the protein by pepsindigestion under quench conditions and following the mass increase of theresulting peptides.

One use of HX-MS is to probe for sites involved in molecularinteractions by identifying regions of reduced hydrogen exchange uponprotein-protein complex formation. Usually, binding interfaces will berevealed by marked reductions in hydrogen exchange due to stericexclusion of solvent. Protein-protein complex formation may be detectedby HX-MS simply by measuring the total amount of deuterium incorporatedin either protein members in the presence and absence of the respectivebinding partner as a function of time. The HX-MS technique uses thenative components, ie protein and antibody or Fab fragment, and isperformed in solution. Thus HX-MS provides the possibility for mimickingthe in vivo conditions (for a recent review on the HX-MS technology, seeWales and Engen, Mass Spectrom. Rev. 25, 158 (2006)).

Protein Batches Used were:

hDR3: Full extracellular domain oh hDR3 fused to Fc (SEQ ID NO 7). Thismolecule is dimeric with respect to hDR3 due to the dimerization of theFc. An expression batch also contains oligomerized version of theprotein, but only the purified dimeric fraction was used for HX-MSexperiments.

Fab molecules: 0120, 0130, 0143, 0148, 0163, 0228, 0230 and 0231 (seetable 29). All proteins were buffer exchanged into PBS pH 7.4 beforeexperiments.

Instrumentation and Data Recording

The HX experiments were performed on a nanoACQUITY UPLC System with HDXTechnology (Waters Inc.) coupled to a Synapt G2 mass spectrometer(Waters Inc.). The Waters HDX system contained a Leap robot (H/D-x PAL;Waters Inc.) operated by the LeapShell software (Leap TechnologiesInc/Waters Inc.), which performed initiation of the deuterium exchangereaction, reaction time control, quench reaction, injection onto theUPLC system and digestion time control. The Leap robot was equipped withtwo temperature controlled stacks maintained at 20° C. for bufferstorage and HX reactions and maintained at 2° C. for storage of proteinand quench solution, respectively. The Waters HDX system furthermorecontained a temperature controlled chamber holding the pre- andanalytical columns, and the LC tubing and switching valves at 1° C. Aseparately temperature controlled chamber holds the pepsin column at 25°C. For the inline pepsin digestion, 100 μL quenched sample containing200 μmol hDR3 was loaded and passed over a Poroszyme® Immobilized PepsinCartridge (2.1×30 mm (Applied Biosystems)) placed at 25° C. using aisocratic flow rate of 100 μL/min (0.1% formic acid:CH₃CN 95:5). Theresulting peptides were trapped and desalted on a VanGuard pre-columnBEH C18 1.7 μm (2.1×5 mm (Waters Inc.)). Subsequently, the valves wereswitched to place the pre-column inline with the analytical column,UPLC-BEH C18 1.7 μm (1×100 mm (Waters Inc.)), and the peptides separatedusing a 9 min gradient of 10-40% B delivered at 200 μl/min from thenanoAQUITY UPLC system (Waters Inc.). The mobile phases consisted of A:0.1% formic acid and B: 0.1% formic acid in CH₃CN. The ESI MS data, andthe separate elevated energy (MS^(E)) experiments were acquired inpositive ion mode using a Synapt G2 mass spectrometer (Waters Inc.).Leucine-enkephalin was used as the lock mass ([M+H]⁺ion at m/z 556.2771)and data was collected in continuum mode (For further description, seeAndersen and Faber, Int. J. Mass Spec., 302, 139-148 (2011)).

Data Analysis

Peptic peptides were identified in separate experiments using standardMS^(E) methods where the peptides and fragments are further alignedutilizing the ion mobility properties of the Synapt G2 (Waters Inc.).MS^(E) data were processed using ProteinLynx Global Server versionversion 2.5 (Waters Inc.). The HX-MS raw data files were processed inthe DynamX software (Waters Inc.). DynamX automatically performs thelock mass-correction and deuterium incorporation determination, i.e.,centroid determination of deuterated peptides. Furthermore, all peptideswere inspected manually to ensure correct peak and deuterationassignment by the software.

Epitope Mapping Experiment

Amide hydrogen/deuterium exchange (HX) was initiated by a 7-folddilution of hDR3 in the presence or absence of Fabs 0120, 0130, 0143,0148, 0163, 0228, 0230 or 0231 into the corresponding deuterated buffer(i.e. PBS prepared in D₂O, 96% D₂O final, pH 7.4 (uncorrected value)).All HX reactions were carried out at 20° C. and contained 2 μM hDR3 inthe absence or presence of 10 μM Fab thus giving a 5 fold molar excessof Fab. At appropriate time intervals ranging from 10 sec to 3000 sec,50 μl aliquots of the HX reaction were quenched by 50 μl ice-coldquenching buffer (1.35M TCEP) resulting in a final pH of 2.5(uncorrected value).

Results and Discussion

The HX time-course of 20 peptic peptides, covering 75% of the primarystructure of hDR3 extracellular domain were monitored in the absence orpresence of 0120, 0130, 0143, 0148, 0163, 0228, 0230 or 0231 Fab for 10to 3000 sec. hDR3 contains two N-glycosylation sites at positions N67and N106 and the peptide map thus has gaps in these regions.

The observed exchange pattern in the early time points (<300 sec) in thepresence or absence of Fabs 0120, 0130, 0143, 0148, 0163, 0228, 0230 or0231 can be divided into two different groups: One group of peptidesdisplay an exchange pattern that is unaffected by the binding of Fabs.In contrast, another group of peptides in hDR3 show protection fromexchange upon Fab binding (table 29). Observed exchange protection in apeptide is indicative of this region being involved in Fab binding. Thusthe epitope is partly or maybe even fully located within the regiondefined by the specific peptides. However, since the resolution of HX-MSis based on peptic peptide, exchange protection within a given regiondoes not means that every residue within the region necessarily isinvolved in Fab binding.

Epitope Mapping of 0148 and 0163

Fabs 0148 and 0163 represent Fab molecules that bind hDR3 but do notblock TL1A binding. These Fab molecules were included in the study forcontrol. Epitope signal was observed in region P140-L153 in CRD3 for Fab0148 (table 29). The current experiment was performed with 5× surplus ofFab, however HX-MS experiments using equimolar rations of 0148 to hDR3confirms that this Fab can be mapped under standard equimolarconditions.

Weak epitope signal for Fab 0163 was observed partly in region G163-F169and partly in region Y170-L184 in the CRD4 region.

Epitope Mapping of 0120, 0130, 0143 and 0228

Fab 0228 was mapped in several experiments using 3-5× surplus of Fabmolecule. Epitope signal was observed in regions R32-G54, G37-L45 andF46-A59 in CRD1. However, based on the relative level of exchangeprotection in the peptic peptides and the weak or absent exchangeprotection in region F46-Y56, it is concluded that the epitope for 0228is strongest in region G37-L45 and L57-A59. The region G37-L45 alsocovers the residues found by mutagenesis to be important for 0228/0123binding to hDR3 and the data from these experiments are thus in fullaccordance (Example 12, Table 16). Surprisingly, it was necessary to usea large surplus of 0228 Fab relative to hDR3. Normally, it is sufficientto use equimolar ratio of mAb or Fab when performing epitope mapping byHX-MS. If using equimolar ration of 0228 to hDR3, no epitope signal wasobserved (data not shown) even though 0228 has a very high affinity(Example 11, table 14). These observations points towards the 0228epitop being in a region that is not fully solvent accessible under theexperimental conditions. Thus the low exchange protection in spite of ahigh affinity of 0228 could be a result of the fab molecule having toeither appear on top of—or compete with—hDR3 self-interactions. The hDR3self-interaction may be a consequence of unspecific aggregation or aconsequence of clustering of the extracellular domain as for exampledescribed for other members of the TNFR super-family (Mukai et al.(2010) Sci. Signal. 3, ra83). Furthermore, weak effects could beobserved in the C-terminal region. Since 0228 and 0120 have fullaffinity when binding to a protein containing only CRD1 (Example 11,table 14), the C-terminal effects is most likely a result ofconformational rearrangements upon Fab binding and may be a result ofmovements in the hinge region fusing DR3 to Fc.

From SPR experiments it has been established that 0120, 0130 and 0143all compete with 0228 and with each other for binding to hDR3 (Example11, Table 15). Thus, these Fabs share epitopes either partly or fully.

In agreement, HX-MS on these Fabs also reveal epitope signal in the sameregion G37-L45. However the exchange protection magnitude is weaker.Based on the observations above it can be speculated that in order tohave HX-MS reveal exchange protection in a clustering interface it isneeded to have high surplus of the binding molecule and it should be ahigh affinity interaction.

Epitope Mapping of 0230 and 0231

Epitope mapping on Fab 0230 and 0231 (11H08 from WO2011106707) did notreveal any epitope. Given the observations described above that epitopemapping on hDR3 need a good affinity in order to be successful it can bespeculated that the relatively poorer affinity of these Fabs(WO2011106707) preclude successful epitope mapping by HX-MS.

TABLE 29 HXMS analysis of DR3 yielding epitope information for Fabmolecules. After deuterium exchange reaction. DR3 is digested withpepsin yielding the following peptic peptide regions that were analyzed.Positions N67 and N106 are glycosylated and the peptide map thus hasgaps in these regions. Numbering of DR3 residues follows SEQ ID NO 1.0148 0163 0120 0130 0143 non- non- 0228 Sequence Region blockingblocking blocking blocking blocking blocking R32-G54 CRD1 EX NA NA N NAEX G37-L45 CRD1 W W W N N EX F46-Y56 CRD1 W/N N N N N W/N F46-A59 CRD1EX/W N NA N N EX L71-F78 CRD2 N N N N N N L71-L79 CRD2 N N N N N NL79-E88 CRD2 N N N N N N L79-C89 CRD2 N N N N N N A80-C89 CRD2 N N N N NN V110-W121 CRD2/CRD3 N N N N N N V110-F122 CRD2/CRD3 N N N N N NV123-C130 CRD3 NA N N N N N V131-L142 CRD3 N NA N N N N P140-L153 CRD3 NN N EX N N D143-L153 CRD3 N N N EX N N L154-L166 CRD3/CRD4 N N N N N NL154-F169 CRD3/CRD4 N N N N W N G163-F169 CRD4 N N N N W N Y170-L184CRD4 N/W N N N W N/W Y170-A193 CRD4 N/W N N N W N/W EX: exchangeprotection upon Fab binding indicating epitope region (>0.4 Da). W: Weakexchange protection upon Fab binding (0.2-0.4 Da). N: No exchangeprotection upon Fab binding (<0.2 Da). NA: Not analyzable in respectiveexperiment.

More than one assignment indicates that two or more experiments havegiven slightly different results regarding magnitude of exchangeprotection.

Example 21 Overview of Antibodies Mentioned Herein

Table 30 provides and overview of antibodies mentioned herein and thenaming of them dependent on the different mAb/Fab formats used. A noteon nomenclature: Table 30 uses four-number identification (e.g. 0228).However, different nomenclature can be observed for the same compound.For example compound 0228 is also be named 0227-0000-0228, 0227-0228,00228 or 228. All these nomenclature denotes the same compound. Thisrule applies to all compounds.

TABLE 30 List of anti-DR3 mAb and Fab compounds selected for studies.This table gives the correlation between mAb, Fab and hydridoma clonenumbering. Compounds in the same row have the same CDR regions andoriginate from the same murine mAb, but have been prepared in differentformats. Compound in the same column share the same molecular format,e.g. species or isotype of the compound, but have been prepared fromdifferent murine mAbs. Chimeric Fab. Chimeric Chimeric Murine Fab. Fab.variable Murine Murine domain, Fab variable variable human from domain,domain, IgG4 hybridoma re- papain Re- human human constant purified-combinant cleaved combinant IgG1 IgG4 domain. Humanized Humanizedblocking SEQ Hybridoma murine murine murine murine constant constantHinge Fab, Fab, TL1A ID number mAb mAb mAb Fab domain. domain. Cys ->Ser IgG4 IgG1 binding NO 5F13 0025 0121 0140 0147 0149 0148 No 24, 2527F16 or 0070 or 0106 0110 0118 0125 0122 partially  8, 9 27F16A1 008327F38 0071 no 27F44 or 0072 or 0107 0087 0111 0119 0126 0123 0227, 0169,yes 10, 11, 27F44A2 0084 0228 (S49A 0170, 16, 17 HC), 0171, 0229 (S49A0172, HC, S60A 0173, LC) 0174, 0222, 0219 (S49A HC), 0226 (S49A HC, S60ALC) 28F26 or 0073 or 0108 0088 0112 0120 0127 0124 yes 12, 13 28F26A30085 28F69 0074 no 29F6 0075 no 29F8 0076 or 0116 0089 yes 0086 30F10077 0091 no 44F434 0128 0143 yes 20, 21 44F478 0129 0144 partially45F36 0130 yes 18, 19 45F187 0150 or no 26, 27 0163 45F243 0184 or yes0185 45F284 0131 0145 partially 45F304 0132 0146 no 46F108 0136 yes46F147 or 0135 0151 yes 46F42 46F164 0137 partially 46F172 0138partially 46F186 0139 yes 48F312 0179 no 49F23 0180 partially 50F1 0159yes 50F3 0177 yes 50F62 0186 yes 50F134 0160 yes 50F141 0161 partially50F142 0162 yes 50F189 0178 yes 50F191 0152 yes 22, 23 50F213 0187 yes11H08 0230 (H1L2) yes 14, 15 (WO 0231 (H1L4) 2011106707) aTNP 0158*isotype control, does not bind DR3 *Isotype control - not DR3 binding

Example 22 Binding Experiment

Material and Methods: Buffy Coats were obtained from normal healthyvolunteers from Copenhagen Hospital. CD4+ T cells were isolated throughmagnetic bead separation. Cells were activated with 2 ng/ml IL-12, 50ng/ml IL-18 and 100 ng/ml TL1A (Flag-HIS-TEV-TL1A—produced at NovoNordisk A/S) and cultured for 5 days. On day 5 cells were stained with10, 5, 1, 0.5, 0.1 or 0.0001 μg/ml of the anti-DR3 mAb clones 0072,0073, or 0077, or with Fab fragment clones 0087, 0088 or 0091. Asecondary PE conjugated goat-anti-mouse (H+L) was used for detection.Samples were in duplicate. Staining intensity was evaluated by FACS.

Results: No binding to the cells could be detected for the monoclonalIgG clone 0077, and its corresponding Fab clone 0091. The monoclonal IgGclones 0072 and 0073 showed potent binding to cytokine activated CD4+ Tcells with maximal binding observed at concentrations of 1 ug/ml orlower. The corresponding Fab clones (0087 and 0088) bound with similarpotency to cytokine activated cells, although slightly lower MFI valueswere recorded at binding saturation. The greater observed MFI values offull-length antibody treated compared with Fab treated cells wasexpected since the secondary (detecting) antibody (anti-IgG heavy andlight chain) bound to both Fab and Fc parts of the full-length IgGantibody clones, but only to Fab parts of Fab clones (Table 31).

TABLE 31 Mean fluorescence intensity (MFI) values are shown forIL-12/IL-18/TL1A activated cells on day 5. Cells are gated on singlets,live and CD4+ T cells. Antibodies or Fab Concentration used fragments inassay MFI 0072 1 μg/ml  1300b 0073 1 μg/ml 1300 0077 1 μg/ml  100 0087 1μg/ml  500 0088 1 μg/ml  500 0091 1 μg/ml  100

Example 23 T Cell Proliferation:

CD4+ T cells were isolated from Buffy Coats through magnetic beadseparation using CD4 Rosettesep (Stem cell technologies) and Histopaque(Sigma). T cells (2*10⁵ cells/well in a 96-well plate) were activatedfor 5 days with 2 ng/ml IL-12, 50 ng/ml IL-18 and 100 ng/ml TL1A(Flag-HIS-TEV-TL1A; Novo Nordisk) with and without anti-TL1A (1000ng/ml; MAB7441; RnDSystems) or DR3 mAbs or DR3Fabs (5 or 10 μg/ml) TheDR3 mAbs used were 0072, 0073 and 0077. The DR3Fabs used were 0087, 0088and 0091. All samples were done in triplicate. Cell proliferation wasmeasured by thymidine incorporation. Cells were pulsed with[³H]thymidine after 5 days of activation and harvested 16 h later. Theincorporated thymidine was detected as counts pr minute (cpm) by a TopCounter.

Results: T cell proliferation was increased 3-fold by costimulation withTL1A. This TL1A-dependent increase was blocked by co-incubation with theanti-TL1A neutralizing control antibody. All anti-DR3 mAbs as well asthe Fab 0091 slightly inhibited proliferation at 10 μg/ml, while theFabs 0087 and 0088 completely blocked proliferation even with the lowestconcentration used in the assay. In conclusion, Fabs 0087 and 0088 arevery efficient in downregulating the TL1A-dependent co-stimulation ofCD4-positive T-cell proliferation.

In a similar experiment IL-12 (2 ng/ml), IL-18 (50 ng/ml) and 100 ng/mlTL1A (Flag-His-TEV-TL1A; Novo Nordisk). treated CD4+ T cellsdemonstrated 6-fold increased T cell proliferation compared toIL-12/IL-18 treated T cells. The 0123 and 0124 Fabs totally blocked theinduced T cell proliferation already at a concentration of 0.16 μg/ml ofthe Fab while the corresponding mAb 0072 and 0108 did not affect T cellproliferation. In Table 2 data for the pair of mAb 0072 and thecorresponding Fab 0123 is shown. Proliferation detected by thymidineincorporation is given as counts pr minute (cpm).

TABLE 32 Treatment mAb 0072 Fab 0123 IL-12/IL-18  1629 ± 109 cpm  1629 ±109 cpm IL-12/IL-18 + TL1A 10439 ± 689 cpm 10439 ± 689 cpm IL-12/IL-18 +TL1A +  10434 ± 1132 cpm   5492 ± 1330 cpm 0.00128 μg/ml mAb or FabIL-12/IL-18 + TL1A + 11886 ± 984 cpm  2329 ± 336 cpm 0.16 μg/ml mAb orFab IL-12/IL-18 + TL1A + 10 10139 ± 689 cpm  1619 ± 109 cpm μg/ml mAb orFab The standard deviation is indicated by the number after the ±.

Cytokine Release Affected by Anti-DR3Fabs

CD4+ T cells isolated from Buffy Coats through magnetic bead separationusing CD4 Rosettesep (Stem cell technologies) and Histopaque (Sigma)were stimulated in the absence of TCR activation, with cytokines IL-12(2 ng/ml), IL-18 (50 ng/ml) with and without 100 ng/ml TL1A(Flag-His-TEV-TL1A; Novo Nordisk). Supernatants from activated CD4+ Tcells (2×10⁵ cells/well) were harvested after 48 h and analysed forcytokine release by Bioplex.

The released cytokines IL-6, IFNγ, GM-CSF and TNFα were all inducedafter 48 hours by TL1A by the IL-12/IL-18 treated CD4+ T cells. IL-6,IFNγ, GM-CSF, TNFα were induced 16-fold (p=0.05), 1.2 (p=0.02), 7.5-fold(p=0.04), 4.4 (p=0.0007) by TL1A, compared to IL-12/IL-18 activatedcells. IL-6 (p=0.04), GM-CSF (p=0.04) and TNFα (p=0.0008) were allsignificantly blocked by the anti-DR3Fab 0124, when compared to the DR3binding non-blocking Fab 0091 (Table 33).

TABLE 33 IL-12/IL-18- IL-12/IL-18- IL-12/IL-18- IL-12/IL-18- activatedactivated activated activated CD4+ CD4+ CD4+ CD4+ T cells + T cells +Cytokines T cells T cells + TL1A TL1A + 0124 TL1A + 0091 (mean +/− (mean± (mean ± (mean ± (mean ± SEM) SEM) SEM) SEM) SEM) IL-6 361 ± 145 5981 ±1918 698 ± 282 9268 ± 3564 IFNγ 4207 ± 270  4893 ± 400  4388 ± 195  4945± 516  GM-CSF 45 ± 11 336 ± 101 55 ± 17 437 ± 167 TNFα  23 ± 2.3 102 ±3   24 ± 2.5 121 ± 21  The standard deviation is indicated by the numberafter the ±.

Example 24

Functional Studies Using Lamina Propria Mononuclear Cells Isolated fromIntestinal Biopsies from Crohn's Patients

Lamina Propria Mononuclear Cells (LPMCs) isolated from intestinalbiopsies from Crohn's patients versus non-IBD controls were activatedwith IL-12 (2 ng/ml), IL-18 (50 ng/ml) and 100 ng/ml TL1A(Flag-His-TEV-TL1A; Novo Nordisk) with and without anti-DR3 Fab 0124 andthe non-blocking control 0091.

LPMCs from inflamed Crohn's patients were able to respond toIL-12/IL-18+TL1A by inducing IFNγ and the TL1A-induced level of IFNγ wassignificantly blocked by 0124 (p=0.0001, n=4), Table 34. The DR3Fabs0124 and 0228 furthermore inhibit GM-CSF, TNF-a, and IFNg secretion byIL-12/IL-18 stimulated TL1A treated LPMCs from Crohn's patients.

TABLE 34 IL-12/IL-18- IL-12/IL-18- IL-12/IL-18- activated activatedactivated LPMCs ± LPMCs LPMCs + TL1A TL1A + 0124 Cytokines (mean ± SEM,(mean ± SEM, (mean ± SEM, (mean +/− SEM) n = 4)) n = 4) n = 4) IFNγ 195± 51 577 ± 158 199 ± 53

1. A monovalent antagonistic DR3 antibody, wherein said monovalentantibody blocks binding of DR3 to TL1A, and wherein said monovalentantibody in a bivalent form thereof is an agonistic antibody that blocksbinding of DR3 to TL1A.
 2. A monovalent antibody according to claim 1,wherein the monovalent antibody is not an antibody having the CDRsequences of the 11H08 antibody set forth in WO2011106707 (SEQ ID NOs14+15).
 3. A monovalent antibody according to claim 1, wherein saidmonovalent antibody is conjugated with a lipophilic moiety.
 4. Amonovalent antibody according to claim 3, wherein said lipophilic moietycomprises a —(CH₂)_(n)—CO— fatty acyl group, wherein n is 16-18.
 5. Amonovalent antibody according to claim 3, wherein said lipophilic moietycomprising a —(CH₂)_(n)—CO— fatty acyl group, wherein n is
 15. 6. Amonovalent antibody according to claim 3, wherein said antibody isconjugated to a lipophilic moiety selected from the group consisting offormulas (I), (II), (III), (IV), (V), and (VI):


7. A monovalent antibody according to claim 3, wherein said lipophilicmoiety is attached to a naturally occurring C239 (Kabat numbering)cysteine residue in the heavy chain of the antibody via a hydrophilicspacer.
 8. A monovalent antibody according to claim 1, wherein saidantibody binds to an epitope on DR3, wherein said epitope comprises I43and/or L45 of SEQ ID NO
 1. 9. A monovalent antibody according to claim1, wherein said antibody binds an epitope on DR3, wherein said epitopecomprises at least one of amino acids G37 to L45 and at least one ofamino acids L57 to A59 as set forth in SEQ ID NO
 1. 10. A monovalentantibody according to claim 9, wherein said epitope comprises aminoacids G37 to L45 and amino acids L57 to A59 as set forth in SEQ ID NO 1.11. A monovalent antibody according to claim 1, wherein said ligand is aFab fragment.
 12. A monovalent antibody according to claim 1, whereinsaid monovalent antibody binds DR3 with a dissociation constant of below1 nM.
 13. A monovalent antibody according to claim 1, wherein saidantibody binds to the CDR1 domain of human DR3.
 14. (canceled)
 15. Amonovalent antibody according to claim 1, wherein said antibodycomprises a human frame work, the CDR3 sequence as set forth in SEQ IDNO 16 and the CDR3 sequence as set forth in SEQ ID NO 17 as well as an“S49A” back mutation in the heavy chain.
 16. A monovalent antibodyaccording to claim 15, wherein said antibody comprises the three CDRsequences as set forth in SEQ ID NO 16, and the three CDR sequences asset forth in SEQ ID NO
 17. 17. A monovalent antibody according to claim1, wherein said antibody competes with “0228” monovalent antibody forbinding to human DR3, wherein the amino acid sequence of the 0228 heavychain is at set forth in SEQ ID NO 16 and the amino acid sequence of the0228 light chain is as set forth in SEQ ID NO
 17. 18. A monovalentantibody according to claim 1, wherein said antibody decreases IFN-gamma(IFN-γ) release in synovial fluid cells from RA patients, wherein saidsynovial fluid cells are co-stimulated with TL1A.
 19. A monovalentantibody according to claim 1, wherein said antibody decreases releaseof one or more cytokines in Lamina Propria Mononuclear Cells (LPMCs)from intestinal biopsies from CD patients, wherein said cytokines areselected from the list consisting of: TNF-α, IL-6, GM-CSF, and IFN-gamma(IFN-γ), and wherein said LPMCs are co-stimulated with TL1A, 1L-12, andIL-18.
 20. A monovalent antibody according to claim 1, wherein saidantibody decreases release of one or more cytokines in CD4+ T cells,wherein said cytokines are selected from the list consisting of: TNF-α,IL-6, GM-CSF, and IFN-gamma (IFN-γ), and wherein said T cells areco-stimulated by TL1A.
 21. A monovalent antibody according to claim 1,wherein the antibody is an IgG4 type antibody.
 22. A pharmaceuticalcomposition comprising an antibody according to claim
 1. 23. A method oftreating an inflammatory disease comprising administering a monovalentantibody according to claim 1 to a patient in need thereof. 24.(canceled)
 25. (canceled)
 26. The method of treating an inflammatorydisease of claim 23, wherein the inflammatory disease is Crohn's Disease(CD).
 27. The method of treating an inflammatory disease of claim 23,wherein the inflammatory disease is rheumatoid arthritis (RA).